Quinazolinone, quinolone and related analogs as sirtuin modulators

ABSTRACT

Provided herein are novel sirtuin-modulating compounds and methods of use thereof. The sirtuin-modulating compounds may be used for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders that would benefit from increased mitochondrial activity. Also provided are compositions comprising a sirtuin-modulating compound in combination with another therapeutic agent.

REFERENCE TO RELATED APPLICATION

This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2009/058868, filed Sep. 29, 2009, which claims the benefit of U.S. Provisional Application No. 61/194,576, filed Sep. 29, 2008, the disclosures of which are incorporated herein by reference thereto. International Application No. PCT/US2009/058868 was published under PCT Article 21(2) in English.

BACKGROUND

The Silent Information Regulator (SIR) family of genes represents a highly conserved group of genes present in the genomes of organisms ranging from archaebacteria to eukaryotes. The encoded SIR proteins are involved in diverse processes from regulation of gene silencing to DNA repair. The proteins encoded by members of the SIR gene family show high sequence conservation in a 250 amino acid core domain. A well-characterized gene in this family is S. cerevisiae SIR2, which is involved in silencing HM loci that contain information specifying yeast mating type, telomere position effects and cell aging. The yeast Sir2 protein belongs to a family of histone deacetylases. The Sir2 homolog, CobB, in Salmonella typhimurium, functions as an NAD (nicotinamide adenine dinucleotide)-dependent ADP-ribosyl transferase.

The Sir2 protein is a class III deacetylase which uses NAD as a cosubstrate. Unlike other deacetylases, many of which are involved in gene silencing, Sir2 is insensitive to class I and II histone deacetylase inhibitors like trichostatin A (TSA).

Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NAD hydrolysis, producing nicotinamide and a novel acetyl-ADP ribose compound. The NAD-dependent deacetylase activity of Sir2 is essential for its functions which can connect its biological role with cellular metabolism in yeast. Mammalian Sir2 homologs have NAD-dependent histone deacetylase activity.

Biochemical studies have shown that Sir2 can readily deacetylate the amino-terminal tails of histones H3 and H4, resulting in the formation of 1-O-acetyl-ADP-ribose and nicotinamide. Strains with additional copies of SIR2 display increased rDNA silencing and a 30% longer life span. It has recently been shown that additional copies of the C. elegans SIR2 homolog, sir-2.1, and the D. melanogaster dSir2 gene greatly extend life span in those organisms. This implies that the SIR2-dependent regulatory pathway for aging arose early in evolution and has been well conserved. Today, Sir2 genes are believed to have evolved to enhance an organism's health and stress resistance to increase its chance of surviving adversity.

In humans, there are seven Sir2-like genes (SIRT1-SIRT7) that share the conserved catalytic domain of Sir2. SIRT1 is a nuclear protein with the highest degree of sequence similarity to Sir2. SIRT1 regulates multiple cellular targets by deacetylation including the tumor suppressor p53, the cellular signaling factor NF-κB, and the FOXO transcription factor.

SIRT3 is a homolog of SIRT1 that is conserved in prokaryotes and eukaryotes. The SIRT3 protein is targeted to the mitochondrial cristae by a unique domain located at the N-terminus. SIRT3 has NAD+-dependent protein deacetylase activity and is ubiquitously expressed, particularly in metabolically active tissues. Upon transfer to the mitochondria, SIRT3 is believed to be cleaved into a smaller, active form by a mitochondrial matrix processing peptidase (MPP).

Caloric restriction has been known for over 70 years to improve the health and extend the lifespan of mammals. Yeast life span, like that of metazoans, is also extended by interventions that resemble caloric restriction, such as low glucose. The discovery that both yeast and flies lacking the SIR2 gene do not live longer when calorically restricted provides evidence that SIR2 genes mediate the beneficial health effects of a restricted calorie diet. Moreover, mutations that reduce the activity of the yeast glucose-responsive cAMP (adenosine 3′,5′-monophosphate)-dependent (PKA) pathway extend life span in wild type cells but not in mutant sir2 strains, demonstrating that SIR2 is likely to be a key downstream component of the caloric restriction pathway.

SUMMARY

Provided herein are novel sirtuin-modulating compounds and methods of use thereof.

In one aspect, the invention provides sirtuin-modulating compounds of Structural Formulas (I) to (V) as are described in detail below.

In another aspect, the invention provides methods for using sirtuin-modulating compounds, or compositions comprising sirtuin-modulating compounds. In certain embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for a variety of therapeutic applications including, for example, increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, and/or flushing, etc. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used for treating a disease or disorder in a subject that would benefit from increased mitochondrial activity, for enhancing muscle performance, for increasing muscle ATP levels, or for treating or preventing muscle tissue damage associated with hypoxia or ischemia. In other embodiments, sirtuin-modulating compounds that decrease the level and/or activity of a sirtuin protein may be used for a variety of therapeutic applications including, for example, increasing cellular sensitivity to stress, increasing apoptosis, treatment of cancer, stimulation of appetite, and/or stimulation of weight gain, etc. As described further below, the methods comprise administering to a subject in need thereof a pharmaceutically effective amount of a sirtuin-modulating compound.

In certain aspects, the sirtuin-modulating compounds may be administered alone or in combination with other compounds, including other sirtuin-modulating compounds, or other therapeutic agents.

DETAILED DESCRIPTION 1. Definitions

As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

The term “bioavailable” when referring to a compound is art-recognized and refers to a form of a compound that allows for it, or a portion of the amount of compound administered, to be absorbed by, incorporated into, or otherwise physiologically available to a subject or patient to whom it is administered.

“Biologically active portion of a sirtuin” refers to a portion of a sirtuin protein having a biological activity, such as the ability to deacetylate. Biologically active portions of a sirtuin may comprise the core domain of sirtuins. Biologically active portions of SIRT1 having GenBank Accession No. NP_036370 that encompass the NAD+ binding domain and the substrate binding domain, for example, may include without limitation, amino acids 62-293 of GenBank Accession No. NP_036370, which are encoded by nucleotides 237 to 932 of GenBank Accession No. NM_012238. Therefore, this region is sometimes referred to as the core domain. Other biologically active portions of SIRT1, also sometimes referred to as core domains, include about amino acids 261 to 447 of GenBank Accession No. NP_036370, which are encoded by nucleotides 834 to 1394 of GenBank Accession No. NM_012238; about amino acids 242 to 493 of GenBank Accession No. NP_036370, which are encoded by nucleotides 777 to 1532 of GenBank Accession No. NM_012238; or about amino acids 254 to 495 of GenBank Accession No. NP_036370, which are encoded by nucleotides 813 to 1538 of GenBank Accession No. NM_012238.

The term “companion animals” refers to cats and dogs. As used herein, the term “dog(s)” denotes any member of the species Canis familiaris, of which there are a large number of different breeds. The term “cat(s)” refers to a feline animal including domestic cats and other members of the family Felidae, genus Felis.

“Diabetes” refers to high blood sugar or ketoacidosis, as well as chronic, general metabolic abnormalities arising from a prolonged high blood sugar status or a decrease in glucose tolerance. “Diabetes” encompasses both the type I and type II (Non Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease. The risk factors for diabetes include the following factors: waistline of more than 40 inches for men or 35 inches for women, blood pressure of 130/85 mmHg or higher, triglycerides above 150 mg/dl, fasting blood glucose greater than 100 mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50 mg/dl in women.

The term “ED₅₀” refers to the art-recognized measure of effective dose. In certain embodiments, ED₅₀ means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations. The term “LD₅₀” refers to the art-recognized measure of lethal dose. In certain embodiments, LD₅₀ means the dose of a drug which is lethal in 50% of test subjects. The term “therapeutic index” is an art-recognized term which refers to the therapeutic index of a drug, defined as LD₅₀/ED₅₀.

The term “hyperinsulinemia” refers to a state in an individual in which the level of insulin in the blood is higher than normal.

The term “insulin resistance” refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.

An “insulin resistance disorder,” as discussed herein, refers to any disease or condition that is caused by or contributed to by insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy, cholesterol related disorders, such as gallstones, cholecystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and bone loss, e.g. osteoporosis in particular.

The term “livestock animals” refers to domesticated quadrupeds, which includes those being raised for meat and various byproducts, e.g., a bovine animal including cattle and other members of the genus Bos, a porcine animal including domestic swine and other members of the genus Sus, an ovine animal including sheep and other members of the genus Ovis, domestic goats and other members of the genus Capra; domesticated quadrupeds being raised for specialized tasks such as use as a beast of burden, e.g., an equine animal including domestic horses and other members of the family Equidae, genus Equus.

The term “mammal” is known in the art, and exemplary mammals include humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

“Obese” individuals or individuals suffering from obesity are generally individuals having a body mass index (BMI) of at least 25 or greater. Obesity may or may not be associated with insulin resistance.

The terms “parenteral administration” and “administered parenterally” are art-recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

A “patient”, “subject”, “individual” or “host” refers to either a human or a non-human animal.

The term “pharmaceutically acceptable carrier” is art-recognized and refers to a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be “acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable cancerous growths in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount. Prevention of an infection includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population. Prevention of pain includes, for example, reducing the magnitude of, or alternatively delaying, pain sensations experienced by subjects in a treated population versus an untreated control population.

The term “prophylactic” or “therapeutic” treatment is art-recognized and refers to administration of a drug to a host. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).

The term “pyrogen-free”, with reference to a composition, refers to a composition that does not contain a pyrogen in an amount that would lead to an adverse effect (e.g., irritation, fever, inflammation, diarrhea, respiratory distress, endotoxic shock, etc.) in a subject to which the composition has been administered. For example, the term is meant to encompass compositions that are free of, or substantially free of, an endotoxin such as, for example, a lipopolysaccharide (LPS).

“Replicative lifespan” of a cell refers to the number of daughter cells produced by an individual “mother cell.” “Chronological aging” or “chronological lifespan,” on the other hand, refers to the length of time a population of non-dividing cells remains viable when deprived of nutrients. “Increasing the lifespan of a cell” or “extending the lifespan of a cell,” as applied to cells or organisms, refers to increasing the number of daughter cells produced by one cell; increasing the ability of cells or organisms to cope with stresses and combat damage, e.g., to DNA, proteins; and/or increasing the ability of cells or organisms to survive and exist in a living state for longer under a particular condition, e.g., stress (for example, heatshock, osmotic stress, high energy radiation, chemically-induced stress, DNA damage, inadequate salt level, inadequate nitrogen level, or inadequate nutrient level). Lifespan can be increased by at least about 10%, 20%, 30%, 40%, 50%, 60% or between 20% and 70%, 30% and 60%, 40% and 60% or more using methods described herein.

“Sirtuin-activating compound” refers to a compound that increases the level of a sirtuin protein and/or increases at least one activity of a sirtuin protein. In an exemplary embodiment, a sirtuin-activating compound may increase at least one biological activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100%, or more. Exemplary biological activities of sirtuin proteins include deacetylation, e.g., of histones and p53; extending lifespan; increasing genomic stability; silencing transcription; and controlling the segregation of oxidized proteins between mother and daughter cells.

“Sirtuin protein” refers to a member of the sirtuin deacetylase protein family, or preferably to the sir2 family, which include yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), and human SIRT1 (GenBank Accession No. NM_012238 and NP_036370 (or AF083106)) and SIRT2 (GenBank Accession No. NM_012237, NM_030593, NP_036369, NP_085096, and AF083107) proteins. Other family members include the four additional yeast Sir2-like genes termed “HST genes” (homologues of Sir two) HST1, HST2, HST3 and HST4, and the five other human homologues hSIRT3, hSIRT4, hSIRT5, hSIRT6 and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273). Preferred sirtuins are those that share more similarities with SIRT1, i.e., hSIRT1, and/or Sir2 than with SIRT2, such as those members having at least part of the N-terminal sequence present in SIRT1 and absent in SIRT2 such as SIRT3 has.

“SIRT1 protein” refers to a member of the sir2 family of sirtuin deacetylases. In one embodiment, a SIRT1 protein includes yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), human SIRT1 (GenBank Accession No. NM_012238 or NP_036370 (or AF083106)), and equivalents and fragments thereof. In another embodiment, a SIRT1 protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685. SIRT1 proteins include polypeptides comprising all or a portion of the amino acid sequence set forth in GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685; the amino acid sequence set forth in GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685, and functional fragments thereof. Polypeptides of the invention also include homologs (e.g., orthologs and paralogs), variants, or fragments, of GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685.

As used herein “SIRT2 protein”, “SIRT3 protein”, “SIRT4 protein”, SIRT 5 protein”, “SIRT6 protein”, and “SIRT7 protein” refer to other mammalian, e.g. human, sirtuin deacetylase proteins that are homologous to SIRT1 protein, particularly in the approximately 275 amino acid conserved catalytic domain. For example, “SIRT3 protein” refers to a member of the sirtuin deacetylase protein family that is homologous to SIRT1 protein. In one embodiment, a SIRT3 protein includes human SIRT3 (GenBank Accession No. AAH01042, NP_036371, or NP_001017524) and mouse SIRT3 (GenBank Accession No. NP_071878) proteins, and equivalents and fragments thereof. In another embodiment, a SIRT3 protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in GenBank Accession Nos. AAH01042, NP_036371, NP_001017524, or NP_071878. SIRT3 proteins include polypeptides comprising all or a portion of the amino acid sequence set forth in GenBank Accession AAH01042, NP_036371, NP_001017524, or NP_071878; the amino acid sequence set forth in GenBank Accession Nos. AAH01042, NP_036371, NP_001017524, or NP_071878 with 1 to about 2, 3, 5, 7, 10, 15, 20, 30, 50, 75 or more conservative amino acid substitutions; an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GenBank Accession Nos. AAH01042, NP_036371, NP_001017524, or NP_071878, and functional fragments thereof. Polypeptides of the invention also include homologs (e.g., orthologs and paralogs), variants, or fragments, of GenBank Accession Nos. AAH01042, NP_036371, NP_001017524, or NP_071878. In one embodiment, a SIRT3 protein includes a fragment of SIRT3 protein that is produced by cleavage with a mitochondrial matrix processing peptidase (MPP) and/or a mitochondrial intermediate peptidase (MIP).

The terms “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” are art-recognized and refer to the administration of a subject composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.

The term “therapeutic agent” is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. The term also means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and/or conditions in an animal or human.

The term “therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The phrase “therapeutically-effective amount” means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. For example, certain compositions described herein may be administered in a sufficient amount to produce a desired effect at a reasonable benefit/risk ratio applicable to such treatment.

“Treating” a condition or disease refers to curing as well as ameliorating at least one symptom of the condition or disease.

The term “vision impairment” refers to diminished vision, which is often only partially reversible or irreversible upon treatment (e.g., surgery). Particularly severe vision impairment is termed “blindness” or “vision loss”, which refers to a complete loss of vision, vision worse than 20/200 that cannot be improved with corrective lenses, or a visual field of less than 20 degrees diameter (10 degrees radius).

2. Sirtuin Modulators

In one aspect, the invention provides novel sirtuin-modulating compounds for treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, ocular diseases and disorders, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing, etc. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used for treating a disease or disorder in a subject that would benefit from increased mitochondrial activity, for enhancing muscle performance, for increasing muscle ATP levels, or for treating or preventing muscle tissue damage associated with hypoxia or ischemia. Other compounds disclosed herein may be suitable for use in a pharmaceutical composition and/or one or more methods disclosed herein.

In one embodiment, sirtuin-modulating compounds of the invention are represented by Structural Formula (I):

or a salt thereof, wherein:

each of Z¹, Z², and Z³, is independently selected from N and CR, wherein:

-   -   no more than one of Z¹, Z² and Z³ is N; and     -   R is selected from hydrogen, halo, —OH, —C≡N, fluoro-substituted         C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —S—(C₁-C₂)         fluoro-substituted alkyl, C₁-C₄ alkyl, —O—(C₁-C₄) alkyl,         —S—(C₁-C₄) alkyl and C₃-C₇ cycloalkyl;

- - - - represents an optional bond,

W¹ is selected from —O—, —NH— or —N═, such that when W¹ is —N═, W¹ is bound to C(R²) through a double bond,

W² is —CR⁴═ when W¹ is —NH— or —O—, such that when W² is —CR⁴═, W² is bound to C(R²) through a double bond; and W² is —NR⁴— when W¹ is —N═;

R¹ is selected from a carbocycle and a heterocycle, wherein R¹ is optionally substituted with one to two substitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, ═O, C₃-C₇ cycloalkyl, fluoro-substituted C₁-C₄ alkyl, —O—R³, —S—R³, —(C₁-C₄ alkyl)-N(R³)(R³), —N(R³)(R³), —O—(C₁-C₄ alkyl)-N(R³)(R³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R³)(R³), —C(O)—N(R³)(R³), and —(C₁-C₄ alkyl)-C(O)—N(R³)(R³), and when R¹ is phenyl, R¹ is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, wherein

-   -   each R³ is independently selected from hydrogen, and —C₁-C₄         alkyl; or     -   two R³ are taken together with the nitrogen atom to which they         are bound to form a 4- to 8-membered saturated heterocycle         optionally comprising one additional heteroatom selected from N,         S, S(═O), S(═O)₂, and O, wherein the alkyl is optionally         substituted with one or more —OH, fluoro, —NH₂, —NH(C₁-C₄         alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂         and the saturated heterocycle is optionally substituted at a         carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄         alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂;

R² is selected from a carbocycle and a heterocycle, wherein R² is optionally substituted with one to two substitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R³, —S—R³, —(C₁-C₄ alkyl)-N(R³)(R³), —N(R³)(R³), —O—(C₁-C₄ alkyl)-N(R³)(R³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R³)(R³), —C(O)—N(R³)(R³), —(C₁-C₄ alkyl)-C(O)—N(R³)(R³), —O-phenyl, phenyl, and a second heterocycle, and when R² is phenyl, R² is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, wherein any phenyl or second heterocycle substituent of R² is optionally substituted with halo; —C≡N; C₁-C₄ alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, —NH—(C₁-C₄) alkyl and —N—(C₁-C₄)₂ alkyl;

R⁴ is selected from hydrogen, C₁-C₄ alkyl, C₁-C₄ fluoro-substituted alkyl, C₁-C₄ alkyl-N(R⁷)(R⁷), C₁-C₄ alkyl-C(O)—N(R⁷)(R⁷), C₁-C₄ alkyl-O—R⁷, and C₁-C₄ alkyl-NR⁷—C(O)R⁷, wherein each R⁷ is independently selected from hydrogen and C₁-C₄ alkyl; and

X is selected from —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR⁵-†, —NR⁵—S(O)₂—NH-†, —NH—C(═O)O-†, —OC(═O)NH-†, —NH—C(═O)NR⁵-†, —NR⁵—C(═O)NH-†, —NH—NR⁵-†, —NR⁵—NH-†, —O—NH-†, —NH—O-†, —NH—CR⁵R⁶-†, —CR⁵R⁶—NH-†, —NH—C(═NR⁵)-†, —C(═NR⁵)—NH-†, —C(═O)—NH—CR⁵R⁶-†, —CR⁵R⁶—NH—C(O)-†, —NH—C(═S)—CR⁵R⁶-†, —CR⁵R⁶—C(═S)—NH-†, —NH—S(O)—CR⁵R⁶-†, —CR⁵R⁶—S(O)—NH-†, —NH—S(O)₂—CR⁵R⁶-†, —CR⁵R⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR⁵R⁶-†, —CR⁵R⁶—O—C(═O)—NH-†, —NH—C(═O)—NR⁵—CR⁵R⁶-†, and —CR⁵R⁶—NH—C(═O)—O-†, wherein:

-   -   † represents where X is bound to R¹; and     -   each R⁵ and R⁶ is independently selected from hydrogen, C₁-C₄         alkyl, —CF₃ and (C₁-C₃ alkyl)-CF₃.

In certain embodiments, compounds of Structural Formula (I) are characterized by one or more of following features:

when each of Z¹, Z² and Z³ is CR; W¹ is —O—, W² is —C═, R⁴ is H; and X is —NH—CR⁵R⁶-† or —CR⁵R⁶—NH-†, then R² is other than optionally substituted pyridin-4-yl or unsubstituted morpholin-4-yl;

when each of Z¹, Z² and Z³ is CH; W¹ is —O—; W² is —C═; R⁴ is H or C₁-C₄ alkyl; R² is phenyl; and X is —C(═O)—NH-†, then R¹ is other than 1H-benzimidazol-2-yl, 2,3-dihydro-2-oxo-1H-benzimidazol-5-yl, 4-methylpiperazin-1-yl, 6-(morpholin-4-yl)-pyridin-3-yl, 5-(morpholin-4-yl)isoquinolin-8-yl, 5-chloro-2-(4-methyl-1-piperazinyl)phenyl, 7-fluoro-3,4-dihydro-4-oxo-6-quinazolinyl. 1-methyl-1H-pyrazol-3-yl, 1H-pyrazol-3-yl, tetrazol-5-yl, 5-(1-methylethyl)-1,3,4-thiadiazol-2-yl, 5-(ethylthio)-1,3,4-thiadiazol-2-yl, 5-ethyl-1,3,4-thiadiazol-2-yl, or 4-(pyrrolidin-1-ylmethyl)-thiazol-2-yl; and

when each of Z¹, Z² and Z³ is CH; W¹ is —O—; W² is —C═; R⁴ is H or C₁-C₄ alkyl; R² is phenyl; and X is —C(═O)—NH-†, then R¹ is other than tetrazol-5-yl.

In certain embodiments, the compound of Structural Formula (I) is represented by the following structure:

In certain embodiments, each of Z¹, Z² and Z³ are independently CR. In certain embodiments, one of Z¹, Z², or Z³ is N, such as Z¹ is N, or Z² is N, or Z³ is N. In certain of these embodiments, R is H, such that in certain embodiments, each of Z¹ and Z² are —CH—, each of Z¹ and Z³ are —CH—, each of Z² and Z³ are —CH—, or all of Z¹, Z² and Z³ are —CH—.

In certain embodiments, R is selected from hydrogen, —(C₁-C₄) alkyl-N(R⁷)(R⁷), —(C₁-C₄) alkyl-C(O)—N(R⁷)(R⁷), —(C₂-C₄) alkyl —O—R⁷, and —(C₂-C₄) alkyl-N(R⁷)—C(O)—R⁷. In certain embodiments, R is hydrogen.

In certain embodiments, W¹ is selected from —O—, —NH—, or —N═. In certain embodiments, W² is selected from —NR⁴— or —CR⁴═. In other embodiments, when W¹ is —N═, then W² is selected from —NR⁴— and —CR⁴═. In certain embodiments, when W¹ is —O—, W² is —CR⁴═. In certain embodiments, when W¹ is —NH—, W² is selected from —NR⁴— and —CR⁴═.

In certain embodiments, the compound of Structural Formula (I) is represented by the following structure:

In certain embodiments, the compound of Structural Formula (I) is represented by the following structure:

In certain embodiments R⁴ is selected from hydrogen, —C≡N, C₁-C₄ alkyl, and fluoro-substituted C₁-C₄ alkyl. In certain embodiments, R⁴ is hydrogen. In certain embodiments wherein W¹ is —O— and W² is —CR⁴═, R⁴ is hydrogen. In certain embodiments, when Z¹, Z² and Z³ are —CR—, R⁴ is hydrogen.

In certain embodiments, X is —NH—C(═O)-† or —C(═O)—NH-†. In certain embodiments, X is —NH—C(═O)-†. In an exemplary embodiment, X is —NH—C(═O)-†, Z¹, Z² and Z³ are CR, and R and R⁴ are both H. In certain embodiments, W¹ is —O— and W² is —CR⁴═, Z¹, Z² and Z³ are all CR, and R and R⁴ are both H, and X is —NH—C(═O)-†.

In certain embodiments, the compound of Structural Formula (I) is represented by the following structure:

In certain embodiments, R¹ is selected from heterocycles (e.g., heteroaryls) comprising one or more heteroatoms selected from N, O and S. In particular embodiments, R¹ is selected from heterocycles (e.g., heteroaryls) comprising one or two nitrogens. In particular embodiments, R¹ is selected from heterocycles (e.g., heteroaryls) comprising up to three heteroatoms selected from S and N. In other embodiments, R¹ is selected from heterocycles (e.g., heteroaryls) comprising up to three heteroatoms selected from O and N.

Examples of R¹ include:

In certain such embodiments, R¹ is selected from:

In the embodiments above, R¹ is optionally substituted by 1 or 2 substituents independently selected from halo, (C₁-C₄) alkyl and ═O. In certain embodiments, R¹ is thiazole or pyrazine optionally substituted with one or more substituents selected from halo and (C₁-C₄) alkyl. In certain embodiments, R¹ is optionally substituted thiazole. In other embodiments, R¹ is optionally substituted pyrazine and X is —NH—C(═O)-†.

In certain embodiments, R² is selected from aryl and heteroaryl. In certain embodiments, R² is optionally substituted with one to two substituents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₁-C₂ fluoro-substituted alkyl, —OR⁸ wherein R⁸ is alkyl optionally substituted with one or more halo substituents. In certain embodiments, R² is phenyl optionally substituted by one or more substituents independently selected from —Cl, —Br, —F, —C≡N, —CF₃ and —OCF₃.

Examples of R² include:

In particular embodiments, R² is meta-substituted relative to the attachment of R² to the rest of the compound, and wherein R² is optionally further substituted as described above. In certain embodiments, R² is selected from:

In certain embodiments, Z¹, Z² and Z³ are each independently selected from CR, W¹ is selected from —O—, —N═ and —N, W² is —CR⁴═ or —NR⁴—, X is —NH—C(═O)-†, R¹ is optionally substituted thiazole or pyrazole and R² is optionally substituted phenyl. In particular embodiments, R is H.

In certain embodiments, sirtuin-modulating compounds of the invention are represented by Structural Formula (I)

or a salt thereof, wherein W¹, W², R¹, R⁴, Z¹, Z², and Z³ are as previously defined and:

- - - - represents an optional bond,

R² is selected from a carbocycle and a heterocycle, wherein R² is optionally substituted with one to two substitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R³, —S—R³, —(C₁-C₄ alkyl)-N(R³)(R³), —N(R³)(R³), —O—(C₁-C₄ alkyl)-N(R³)(R³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R³)(R³), —C(O)—N(R³)(R³), —(C₁-C₄ alkyl)-C(O)—N(R³)(R³), —O-phenyl, phenyl, and a second heterocycle, and when R² is phenyl, R² is substituted with at least one substituent selected from: halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R³, —S—R³, —(C₁-C₄ alkyl)-N(R³)(R³), —N(R³)(R³), —O—(C₁-C₄ alkyl)-N(R³)(R³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R³)(R³), —C(O)—N(R³)(R³), —(C₁-C₄ alkyl)-C(O)—N(R³)(R³), —O-phenyl, phenyl, and a second heterocycle, 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, wherein any phenyl or second heterocycle substituent of R² is optionally substituted with halo; —C≡N; C₁-C₄ alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, —NH—(C₁-C₄) alkyl and —N—(C₁-C₄)₂ alkyl; and

X is selected from —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR⁵-†, —NR⁵—S(O)₂—NH-†, —NH—C(═O)O-†, —OC(═O)NH-†, —NH—C(═O)NR⁵-†, —NR⁵—C(═O)NH-†, —NH—NR⁵-†, —NR⁵—NH-†, —O—NH-†, —NH—O-†, —NH—C(═NR⁵)-†, —C(═NR⁵)—NH-†, —C(═O)—NH—CR⁵R⁶-†, —CR⁵R⁶—NH—C(O)-†, —NH—C(═S)—CR⁵R⁶-†, —CR⁵R⁶—C(═S)—NH-†, —NH—S(O)—CR⁵R⁶-†, —CR⁵R⁶—S(O)—NH-†, —NH—S(O)₂—CR⁵R⁶-†, —CR⁵R⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR⁵R⁶-†, —CR⁵R⁶—O—C(═O)—NH-†, —NH—C(═O)—NR⁵—CR⁵R⁶-†, and —CR⁵R⁶—O—C(═O)—NH-†, wherein:

-   -   † represents where X is bound to R¹; and     -   each R⁵ and R⁶ is independently selected from hydrogen, C₁-C₄         alkyl, —CF₃ and (C₁-C₃ alkyl)-CF₃.

In another embodiment, the invention provides a compound represented by Structural Formula (II):

or a salt thereof wherein:

each of Z¹, Z², and Z³ is independently selected from N and C(R⁹), wherein:

-   -   no more than one of Z¹, Z² and Z³ is N;     -   each R⁹ is independently selected from hydrogen, halo, —C≡N,         fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted         alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, C₁-C₄ alkyl,         —S—(C₁-C₄) alkyl, C₃-C₇ cycloalkyl, —(C₁-C₂) alkyl-N(R¹³)(R¹³),         —O—CH₂CH(OH)CH₂OH, —O—(C₁-C₃) alkyl-N(R¹³)(R¹³), and         —N(R¹³)(R¹³); and

represents a second bond between either W² and C(R¹²) or W¹ and C(R¹²);

W¹ is selected from —O—, —NH— or —N═, and

W²(R¹⁴) is selected from —N(R¹⁴)— and —C(R¹⁴)═, selected such that

-   -   when W¹ is —O—, one of Z¹, Z² and Z³ is N;     -   when W¹ is —O— or —NH—, W²(R¹⁴) is —C(R¹⁴)═ and         represents a second bond between W² and C(R¹²);     -   when W¹ is —N═, W²(R¹⁴) is —N(R¹⁴)— and         represents a second bond between W¹ and C(R¹²);

R¹¹ is selected from a carbocycle and a heterocycle, wherein R¹¹ is optionally substituted with one to two substitutents independently selected from halo, —C≡N, C₁-C₄ alkyl, ═O, C₃-C₇ cycloalkyl, fluoro-substituted C₁-C₄ alkyl, —O—R¹³, —S—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), and —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³), and when R¹¹ is phenyl, R¹¹ is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, O-(saturated heterocycle), fluoro-substituted —O-(saturated heterocycle), or C₁-C₄ alkyl-substituted O-(saturated heterocycle), wherein

each R¹³ is independently selected from hydrogen and —C₁-C₄ alkyl; or two R¹³ are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered saturated heterocycle optionally comprising one additional heteroatom selected from NH, S, S(═O), S(═O)₂, and O, wherein

-   -   when R¹³ is alkyl, the alkyl is optionally substituted with one         or more substituents selected from —OH, fluoro, —NH₂, —NH(C₁-C₄         alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), and —N(CH₂CH₂OCH₃)₂         and     -   when two R¹³ are taken together with the nitrogen atom to which         they are bound to form a 4- to 8-membered saturated heterocycle,         the saturated heterocycle is optionally substituted at any         carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄         alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂,         and optionally substituted at any substitutable nitrogen atom         with —C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, or         —(CH₂)₂—O—CH₃;

R¹² is selected from a carbocycle and a heterocycle other than tetrazolyl, wherein R¹² is optionally substituted with one or more substitutents independently selected from halo, —C≡N₁, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹³, —S—R¹³, —S(O)—R¹³, —S(O)₂—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³), —O-phenyl, phenyl, and a second heterocycle, and when R¹² is phenyl, R¹² is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, or —O-(saturated heterocycle), wherein any phenyl, second heterocycle or saturated heterocycle portion of a substituent of R¹² is optionally substituted with halo; —C≡N; C₁-C₄ alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, —NH—(C₁-C₄) alkyl and —N—(C₁-C₄)₂ alkyl;

R¹⁴ is selected from hydrogen, C₁-C₄ alkyl, C₁-C₄ fluoro-substituted alkyl, C₁-C₄ alkyl-N(R¹³)(R¹³), C₁-C₄ alkyl-C(O)—N(R¹³)(R¹³), C₁-C₄ alkyl-O—R¹³, and C₁-C₄ alkyl-NR¹³—C(O)R¹³; and

X¹ is selected from —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR¹⁵-†, —NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†, —OC(═O)NH-†, —NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†, —NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†, —NH—O-†, —NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH-†, —NH—C(═NR¹⁵)-†, —C(═NR¹⁵)—NH-†, —C(═O)—NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—C(═S)—NH-†, —NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†, —NH—S(O)₂—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—O—C(═O)—NH-†, —NH—C(═O)—NR¹⁵—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†, and —CR¹⁵R¹⁶—NH—C(═O)—O-†, wherein:

-   -   † represents where X¹ is bound to R¹¹; and     -   each R¹⁵ and R¹⁶ is independently selected from hydrogen, C₁-C₄         alkyl, —CF₃ and (C₁-C₃ alkyl)-CF₃.

In certain embodiments, compounds of Structural Formula (II) are characterized by one or more of the following features:

the compound of Structural Formula (II) is represented by Structural Formula (III), (IV) or (V):

X¹ is selected from —NH—C(═O)-†, —C(═O)—NH-†, —NH—C(═S)-†, —C(═S)—NH-†, —NH—S(═O)-†, —S(═O)—NH-†, —S(═O)₂—NH-†, —NH—S(═O)₂-†, —NH—S(O)₂—NR¹⁵-†, —NR¹⁵—S(O)₂—NH-†, —NH—C(═O)O-†, —OC(═O)NH-†, —NH—C(═O)NR¹⁵-†, —NR¹⁵—C(═O)NH-†, —NH—NR¹⁵-†, —NR¹⁵—NH-†, —O—NH-†, —NH—O-†, —NH—C(═NR¹⁵)-†, —C(═NR¹⁵)—NH-†, —C(═O)—NH—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—NH—C(O)-†, —NH—C(═S)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—C(═S)—NH-†, —NH—S(O)—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)—NH-†, —NH—S(O)₂—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—S(O)₂—NH-†, —NH—C(═O)—O—CR¹⁵R¹⁶-†, —CR¹⁵R¹⁶—O—C(═O)—NH-†, —NH—C(═O)—NR¹⁵—CR¹⁵R¹⁶-†, —NH—C(═O)—CR¹⁵R¹⁶-†, and —CR¹⁵R¹⁶—NH—C(═O)—O-†;

X¹ is selected from —NH—C(O)-†, and —C(O)—NH-†;

R¹¹ is selected from:

R¹¹ is optionally substituted with one or two substituents independently selected from halo, C₁-C₄ alkyl, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), ═O, —N(R¹³)(R¹³), and —O—R¹³;

R¹¹ is selected from:

R¹¹ is selected from:

R¹² is selected from:

R¹² is optionally substituted with one or more groups independently selected from halo, C₁-C₄ alkyl, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), C₁-C₂ fluoro-substituted alkyl, —O—R¹³, —SO₂—R¹³, —N(R¹³)(R¹³), and —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³);

R¹² is selected from:

R¹² is selected from

R¹² is selected from a carbocycle and a heterocycle having from 1 to 3 heteroatoms;

R¹² is selected from a carbocycle and a heterocycle having from 0 to 3 nitrogens;

R¹² is bound to the rest of the molecule through a ring carbon atom;

R¹² is optionally substituted with one or two substituents;

when R¹² is phenyl, R¹² is substituted with at least one substituent;

W²(R¹⁴) is N(R¹⁴)—, and R¹⁴ is selected from hydrogen and —(C₁-C₄) alkyl;

W²(R¹⁴) is —C(R¹⁴)— and R¹⁴ is hydrogen;

each of Z¹, Z², and Z³ is C(R⁹); and

each of Z¹, Z², and Z³ is —CH═.

Compounds of the invention, including novel compounds of the invention, can also be used in the methods described herein.

The compounds and salts thereof described herein also include their corresponding hydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate) and solvates. Suitable solvents for preparation of solvates and hydrates can generally be selected by a skilled artisan.

The compounds and salts thereof can be present in amorphous or crystalline (including co-crystalline and polymorph) forms.

Sirtuin-modulating compounds of the invention advantageously modulate the level and/or activity of a sirtuin protein, particularly the deacetylase activity of the sirtuin protein.

Separately or in addition to the above properties, certain sirtuin-modulating compounds of the invention do not substantially have one or more of the following activities inhibition of PI3-kinase, inhibition of aldoreductase, inhibition of tyrosine kinase, transactivation of EGFR tyrosine kinase, coronary dilation, or spasmolytic activity, at concentrations of the compound that are effective for modulating the deacetylation activity of a sirtuin protein (e.g., such as a SIRT1 and/or a SIRT3 protein).

Carbocyclic includes 5-7 membered monocyclic and 8-12 membered bicyclic rings wherein the monocyclic or bicyclic rings are selected from saturated, unsaturated and aromatic. A carbocycle is optionally substituted with one or more substituents such as halo, —C≡N, C₁-C₃ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₃) alkyl, —S—(C₁-C₃) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, hydroxyl, amino, —NH—(C₁-C₃) alkyl and —N—(C₁-C₃)₂ alkyl. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl and naphthyl.

Heterocyclic includes 4-7 membered monocyclic and 8-12 membered bicyclic rings comprising one or more heteroatoms selected from, for example, N, O, and S atoms. In certain embodiments, the heterocyclic group is selected from saturated, unsaturated or aromatic. A heterocycle is optionally substituted with one or more substituents such as halo, —C≡N, C₁-C₃ alkyl, C₁-C₂ fluoro-substituted alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₃) alkyl, —S—(C₁-C₃) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, hydroxyl, amino, —NH—(C₁-C₃) alkyl and —N—(C₁-C₃)₂ alkyl.

Monocyclic rings include 5-7 membered aryl or heteroaryl, 3-7 membered cycloalkyl, and 5-7 membered non-aromatic heterocyclyl. Monocyclic rings are optionally substituted with one or more substituents such as halo, cyano, lower alkoxy, lower alkyl, hydroxyl, amino, lower alkylamino and lower dialkylamino. Exemplary monocyclic groups include substituted or unsubstituted heterocycles or carbocycles such as thiazolyl, oxazolyl, oxazinyl, thiazinyl, dithianyl, dioxanyl, isoxazolyl, isothiazolyl, triazolyl, furanyl, tetrahydrofuranyl, dihydrofuranyl, pyranyl, tetrazolyl, pyrazolyl, pyrazinyl, pyridazinyl, imidazolyl, pyridinyl, pyrrolyl, dihydropyrrolyl, pyrrolidinyl, piperidinyl, piperazinyl, pyrimidinyl, morpholinyl, tetrahydrothiophenyl, thiophenyl, cyclohexyl, cyclopentyl, cyclopropyl, cyclobutyl, cycloheptanyl, azetidinyl, oxetanyl, thiiranyl, oxiranyl, aziridinyl, and thiomorpholinyl.

Aromatic (aryl) groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl.

Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuryl, indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.

Azabicyclo refers to a bicyclic molecule that contains a nitrogen atom in the ring skeleton. The two rings of the bicycle may be fused, at two mutually bonded atoms, e.g., indole, across a sequence of atoms, e.g., azabicyclo[2.2.1]heptane, or at a single atom, e.g., spirocycle.

Bridged azabicyclo refers to a bicyclic molecule that contains a nitrogen atom and two fused rings wherein the fusion occurs across a sequence of atoms, i.e., bridgehead atoms. Bridged bicyclo compounds comprise at least one bridge of one or more atoms connecting two bridgehead atoms.

Fluoro-substituted includes from one fluoro substituent up to per-fluoro-substitution. Exemplary fluoro-substituted C₁-C₂ alkyl includes —CFH₂, CF₂H, —CF₃, —CH₂CH₂F, —CH₂CHF₂, —CHFCH₃, and —CF₂CHF₂. Per-fluoro-substituted C₁-C₂ alkyl, for example, includes —CF₃ and —CF₂CF₃.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. As used herein, the term “stable” refers to compounds that possess stability sufficient to allow manufacture and that maintain the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein.

The compounds disclosed herein also include partially and fully deuterated variants. In certain embodiments, deuterated variants may be used for kinetic studies. One of ordinary skill in the art can select the sites at which such deuterium atoms are present.

Also included in the present invention are salts, particularly pharmaceutically acceptable salts, of the sirtuin-modulating compounds described herein. The compounds of the present invention that possess a sufficiently acidic, a sufficiently basic, or both functional groups, can react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Alternatively, compounds that are inherently charged, such as those with a quaternary nitrogen, can form a salt with an appropriate counterion (e.g., a halide such as bromide, chloride, or fluoride, particularly bromide).

Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

According to another embodiment, the present invention provides methods of producing the above-defined sirtuin-modulating compounds. The compounds may be synthesized using conventional techniques. Advantageously, these compounds are conveniently synthesized from readily available starting materials.

Synthetic chemistry transformations and methodologies useful in synthesizing the sirtuin-modulating compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed. (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis (1995).

In an exemplary embodiment, a sirtuin-modulating compound may traverse the cytoplasmic membrane of a cell. For example, a compound may have a cell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.

Sirtuin-modulating compounds described herein may also have one or more of the following characteristics: the compound may be essentially non-toxic to a cell or subject; the sirtuin-modulating compound may be an organic molecule or a small molecule of 2000 amu or less, 1000 amu or less; a compound may have a half-life under normal atmospheric conditions of at least about 30 days, 60 days, 120 days, 6 months or 1 year; the compound may have a half-life in solution of at least about 30 days, 60 days, 120 days, 6 months or 1 year; a sirtuin-modulating compound may be more stable in solution than resveratrol by at least a factor of about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 fold or 100 fold; a sirtuin-modulating compound may promote deacetylation of the DNA repair factor Ku70; a sirtuin-modulating compound may promote deacetylation of RelA/p65; a compound may increase general turnover rates and enhance the sensitivity of cells to TNF-induced apoptosis.

In certain embodiments, a sirtuin-modulating compound does not have any substantial ability to inhibit a histone deacetylase (HDACs) class I, a HDAC class II, or HDACs I and II, at concentrations (e.g., in vivo) effective for modulating the deacetylase activity of the sirtuin. For instance, in preferred embodiments the sirtuin-modulating compound is a sirtuin-activating compound and is chosen to have an EC₅₀ for activating sirtuin deacetylase activity that is at least 5 fold less than the EC₅₀ for inhibition of an HDAC I and/or HDAC II, and even more preferably at least 10 fold, 100 fold or even 1000 fold less. Methods for assaying HDAC I and/or HDAC II activity are well known in the art and kits to perform such assays may be purchased commercially. See e.g., BioVision, Inc. (Mountain View, Calif.; world wide web at biovision.com) and Thomas Scientific (Swedesboro, N.J.; world wide web at tomassci.com).

In certain embodiments, a sirtuin-modulating compound does not have any substantial ability to modulate sirtuin homologs. In one embodiment, an activator of a human sirtuin protein may not have any substantial ability to activate a sirtuin protein from lower eukaryotes, particularly yeast or human pathogens, at concentrations (e.g., in vivo) effective for activating the deacetylase activity of human sirtuin. For example, a sirtuin-activating compound may be chosen to have an EC₅₀ for activating a human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity that is at least 5 fold less than the EC₅₀ for activating a yeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), and even more preferably at least 10 fold, 100 fold or even 1000 fold less. In another embodiment, an inhibitor of a sirtuin protein from lower eukaryotes, particularly yeast or human pathogens, does not have any substantial ability to inhibit a sirtuin protein from humans at concentrations (e.g., in vivo) effective for inhibiting the deacetylase activity of a sirtuin protein from a lower eukaryote. For example, a sirtuin-inhibiting compound may be chosen to have an IC₅₀ for inhibiting a human sirtuin, such as SIRT1 and/or SIRT3, deacetylase activity that is at least 5 fold less than the IC₅₀ for inhibiting a yeast sirtuin, such as Sir2 (such as Candida, S. cerevisiae, etc.), and even more preferably at least 10 fold, 100 fold or even 1000 fold less.

In certain embodiments, a sirtuin-modulating compound may have the ability to modulate one or more sirtuin protein homologs, such as, for example, one or more of human SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7. In one embodiment, a sirtuin-modulating compound has the ability to modulate both a SIRT1 and a SIRT3 protein.

In other embodiments, a SIRT1 modulator does not have any substantial ability to modulate other sirtuin protein homologs, such as, for example, one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, at concentrations (e.g., in vivo) effective for modulating the deacetylase activity of human SIRT1. For example, a sirtuin-modulating compound may be chosen to have an ED₅₀ for modulating human SIRT1 deacetylase activity that is at least 5 fold less than the ED₅₀ for modulating one or more of human SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7, and even more preferably at least 10 fold, 100 fold or even 1000 fold less. In one embodiment, a SIRT1 modulator does not have any substantial ability to modulate a SIRT3 protein.

In other embodiments, a SIRT3 modulator does not have any substantial ability to modulate other sirtuin protein homologs, such as, for example, one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, or SIRT7, at concentrations (e.g., in vivo) effective for modulating the deacetylase activity of human SIRT3. For example, a sirtuin-modulating compound may be chosen to have an ED₅₀ for modulating human SIRT3 deacetylase activity that is at least 5 fold less than the ED₅₀ for modulating one or more of human SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, or SIRT7, and even more preferably at least 10 fold, 100 fold or even 1000 fold less. In one embodiment, a SIRT3 modulator does not have any substantial ability to modulate a SIRT1 protein.

In certain embodiments, a sirtuin-modulating compound may have a binding affinity for a sirtuin protein of about 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M or less. A sirtuin-modulating compound may reduce (activator) or increase (inhibitor) the apparent Km of a sirtuin protein for its substrate or NAD+(or other cofactor) by a factor of at least about 2, 3, 4, 5, 10, 20, 30, 50 or 100. In certain embodiments, Km values are determined using the mass spectrometry assay described herein. Preferred activating compounds reduce the Km of a sirtuin for its substrate or cofactor to a greater extent than caused by resveratrol at a similar concentration or reduce the Km of a sirtuin for its substrate or cofactor similar to that caused by resveratrol at a lower concentration. A sirtuin-modulating compound may increase the Vmax of a sirtuin protein by a factor of at least about 2, 3, 4, 5, 10, 20, 30, 50 or 100. A sirtuin-modulating compound may have an ED50 for modulating the deacetylase activity of a SIRT1 and/or SIRT3 protein of less than about 1 nM, less than about 10 nM, less than about 100 nM, less than about 1 μM, less than about 10 μM, less than about 100 μM, or from about 1-10 nM, from about 10-100 nM, from about 0.1-1 μM, from about 1-10 μM or from about 10-100 μM. A sirtuin-modulating compound may modulate the deacetylase activity of a SIRT1 and/or SIRT3 protein by a factor of at least about 5, 10, 20, 30, 50, or 100, as measured in a cellular assay or in a cell based assay. A sirtuin-activating compound may cause at least about 10%, 30%, 50%, 80%, 2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater induction of the deacetylase activity of a sirtuin protein relative to the same concentration of resveratrol. A sirtuin-modulating compound may have an ED50 for modulating SIRT5 that is at least about 10 fold, 20 fold, 30 fold, 50 fold greater than that for modulating SIRT1 and/or SIRT3.

3. Exemplary Uses

In certain aspects, the invention provides methods for modulating the level and/or activity of a sirtuin protein and methods of use thereof.

In certain embodiments, the invention provides methods for using sirtuin-modulating compounds wherein the sirtuin-modulating compounds activate a sirtuin protein, e.g., increase the level and/or activity of a sirtuin protein. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be useful for a variety of therapeutic applications including, for example, increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing, etc. The methods comprise administering to a subject in need thereof a pharmaceutically effective amount of a sirtuin-modulating compound, e.g., a sirtuin-activating compound.

Without wishing to be bound by theory, it is believed that activators of the instant invention may interact with a sirtuin at the same location within the sirtuin protein (e.g., active site or site affecting the Km or Vmax of the active site). It is believed that this is the reason why certain classes of sirtuin activators and inhibitors can have substantial structural similarity.

In certain embodiments, the sirtuin-modulating compounds described herein may be taken alone or in combination with other compounds. In one embodiment, a mixture of two or more sirtuin-modulating compounds may be administered to a subject in need thereof. In another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered with one or more of the following compounds: resveratrol, butein, fisetin, piceatannol, or quercetin. In an exemplary embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered in combination with nicotinic acid. In another embodiment, a sirtuin-modulating compound that decreases the level and/or activity of a sirtuin protein may be administered with one or more of the following compounds: nicotinamide (NAM), suramin; NF023 (a G-protein antagonist); NF279 (a purinergic receptor antagonist); Trolox (6-hydroxy-2,5,7,8,tetramethylchroman-2-carboxylic acid); (−)-epigallocatechin (hydroxy on sites 3,5,7,3′,4′, 5′); (−)-epigallocatechin gallate (Hydroxy sites 5,7,3′,4′,5′ and gallate ester on 3); cyanidin chloride (3,5,7,3′,4′-pentahydroxyflavylium chloride); delphinidin chloride (3,5,7,3′,4′,5′-hexahydroxyflavylium chloride); myricetin (cannabiscetin; 3,5,7,3′,4′,5′-hexahydroxyflavone); 3,7,3′,4′,5′-pentahydroxyflavone; gossypetin (3,5,7,8,3′,4′-hexahydroxyflavone), sirtinol; and splitomicin. In yet another embodiment, one or more sirtuin-modulating compounds may be administered with one or more therapeutic agents for the treatment or prevention of various diseases, including, for example, cancer, diabetes, neurodegenerative diseases, cardiovascular disease, blood clotting, inflammation, flushing, obesity, aging, stress, etc. In various embodiments, combination therapies comprising a sirtuin-modulating compound may refer to (1) pharmaceutical compositions that comprise one or more sirtuin-modulating compounds in combination with one or more therapeutic agents (e.g., one or more therapeutic agents described herein); and (2) co-administration of one or more sirtuin-modulating compounds with one or more therapeutic agents wherein the sirtuin-modulating compound and therapeutic agent have not been formulated in the same compositions (but may be present within the same kit or package, such as a blister pack or other multi-chamber package; connected, separately sealed containers (e.g., foil pouches) that can be separated by the user; or a kit where the sirtuin modulating compound(s) and other therapeutic agent(s) are in separate vessels). When using separate formulations, the sirtuin-modulating compound may be administered at the same, intermittent, staggered, prior to, subsequent to, or combinations thereof, with the administration of another therapeutic agent.

In certain embodiments, methods for reducing, preventing or treating diseases or disorders using a sirtuin-modulating compound may also comprise increasing the protein level of a sirtuin, such as human SIRT1, SIRT2 and/or SIRT3, or homologs thereof. Increasing protein levels can be achieved by introducing into a cell one or more copies of a nucleic acid that encodes a sirtuin. For example, the level of a sirtuin can be increased in a mammalian cell by introducing into the mammalian cell a nucleic acid encoding the sirtuin, e.g., increasing the level of SIRT1 by introducing a nucleic acid encoding the amino acid sequence set forth in GenBank Accession No. NP_036370 and/or increasing the level of SIRT3 by introducing a nucleic acid encoding the amino acid sequence set forth in GenBank Accession No. AAH01042.

A nucleic acid that is introduced into a cell to increase the protein level of a sirtuin may encode a protein that is at least about 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence of a sirtuin, e.g., SIRT1 and/or SIRT3 protein. For example, the nucleic acid encoding the protein may be at least about 80%, 85%, 90%, 95%, 98%, or 99% identical to a nucleic acid encoding a SIRT1 (e.g. GenBank Accession No. NM_012238) and/or SIRT3 (e.g., GenBank Accession No. BC001042) protein. The nucleic acid may also be a nucleic acid that hybridizes, preferably under stringent hybridization conditions, to a nucleic acid encoding a wild-type sirtuin, e.g., SIRT1 and/or SIRT3 protein. Stringent hybridization conditions may include hybridization and a wash in 0.2×SSC at 65° C. When using a nucleic acid that encodes a protein that is different from a wild-type sirtuin protein, such as a protein that is a fragment of a wild-type sirtuin, the protein is preferably biologically active, e.g., is capable of deacetylation. It is only necessary to express in a cell a portion of the sirtuin that is biologically active. For example, a protein that differs from wild-type SIRT1 having GenBank Accession No. NP_036370, preferably contains the core structure thereof. The core structure sometimes refers to amino acids 62-293 of GenBank Accession No. NP_036370, which are encoded by nucleotides 237 to 932 of GenBank Accession No. NM_012238, which encompasses the NAD binding as well as the substrate binding domains. The core domain of SIRT1 may also refer to about amino acids 261 to 447 of GenBank Accession No. NP_036370, which are encoded by nucleotides 834 to 1394 of GenBank Accession No. NM_012238; to about amino acids 242 to 493 of GenBank Accession No. NP_036370, which are encoded by nucleotides 777 to 1532 of GenBank Accession No. NM_012238; or to about amino acids 254 to 495 of GenBank Accession No. NP_036370, which are encoded by nucleotides 813 to 1538 of GenBank Accession No. NM_012238. Whether a protein retains a biological function, e.g., deacetylation capabilities, can be determined according to methods known in the art.

In certain embodiments, methods for reducing, preventing or treating diseases or disorders using a sirtuin-modulating compound may also comprise decreasing the protein level of a sirtuin, such as human SIRT1, SIRT2 and/or SIRT3, or homologs thereof. Decreasing a sirtuin protein level can be achieved according to methods known in the art. For example, an siRNA, an antisense nucleic acid, or a ribozyme targeted to the sirtuin can be expressed in the cell. A dominant negative sirtuin mutant, e.g., a mutant that is not capable of deacetylating, may also be used. For example, mutant H363Y of SIRT1, described, e.g., in Luo et al. (2001) Cell 107:137 can be used. Alternatively, agents that inhibit transcription can be used.

Methods for modulating sirtuin protein levels also include methods for modulating the transcription of genes encoding sirtuins, methods for stabilizing/destabilizing the corresponding mRNAs, and other methods known in the art.

Aging/Stress

In one embodiment, the invention provides a method extending the lifespan of a cell, extending the proliferative capacity of a cell, slowing aging of a cell, promoting the survival of a cell, delaying cellular senescence in a cell, mimicking the effects of calorie restriction, increasing the resistance of a cell to stress, or preventing apoptosis of a cell, by contacting the cell with a sirtuin-modulating compound of the invention that increases the level and/or activity of a sirtuin protein. In an exemplary embodiment, the methods comprise contacting the cell with a sirtuin-activating compound.

The methods described herein may be used to increase the amount of time that cells, particularly primary cells (i.e., cells obtained from an organism, e.g., a human), may be kept alive in a cell culture. Embryonic stem (ES) cells and pluripotent cells, and cells differentiated therefrom, may also be treated with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein to keep the cells, or progeny thereof, in culture for longer periods of time. Such cells can also be used for transplantation into a subject, e.g., after ex vivo modification.

In one embodiment, cells that are intended to be preserved for long periods of time may be treated with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. The cells may be in suspension (e.g., blood cells, serum, biological growth media, etc.) or in tissues or organs. For example, blood collected from an individual for purposes of transfusion may be treated with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein to preserve the blood cells for longer periods of time. Additionally, blood to be used for forensic purposes may also be preserved using a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. Other cells that may be treated to extend their lifespan or protect against apoptosis include cells for consumption, e.g., cells from non-human mammals (such as meat) or plant cells (such as vegetables).

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be applied during developmental and growth phases in mammals, plants, insects or microorganisms, in order to, e.g., alter, retard or accelerate the developmental and/or growth process.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to treat cells useful for transplantation or cell therapy, including, for example, solid tissue grafts, organ transplants, cell suspensions, stem cells, bone marrow cells, etc. The cells or tissue may be an autograft, an allograft, a syngraft or a xenograft. The cells or tissue may be treated with the sirtuin-modulating compound prior to administration/implantation, concurrently with administration/implantation, and/or post administration/implantation into a subject. The cells or tissue may be treated prior to removal of the cells from the donor individual, ex vivo after removal of the cells or tissue from the donor individual, or post implantation into the recipient. For example, the donor or recipient individual may be treated systemically with a sirtuin-modulating compound or may have a subset of cells/tissue treated locally with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. In certain embodiments, the cells or tissue (or donor/recipient individuals) may additionally be treated with another therapeutic agent useful for prolonging graft survival, such as, for example, an immunosuppressive agent, a cytokine, an angiogenic factor, etc.

In yet other embodiments, cells may be treated with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein in vivo, e.g., to increase their lifespan or prevent apoptosis. For example, skin can be protected from aging (e.g., developing wrinkles, loss of elasticity, etc.) by treating skin or epithelial cells with a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. In an exemplary embodiment, skin is contacted with a pharmaceutical or cosmetic composition comprising a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. Exemplary skin afflictions or skin conditions that may be treated in accordance with the methods described herein include disorders or diseases associated with or caused by inflammation, sun damage or natural aging. For example, the compositions find utility in the prevention or treatment of contact dermatitis (including irritant contact dermatitis and allergic contact dermatitis), atopic dermatitis (also known as allergic eczema), actinic keratosis, keratinization disorders (including eczema), epidermolysis bullosa diseases (including pemphigus), exfoliative dermatitis, seborrheic dermatitis, erythemas (including erythema multiforme and erythema nodosum), damage caused by the sun or other light sources, discoid lupus erythematosus, dermatomyositis, psoriasis, skin cancer and the effects of natural aging. In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for the treatment of wounds and/or burns to promote healing, including, for example, first-, second- or third-degree burns and/or thermal, chemical or electrical burns. The formulations may be administered topically, to the skin or mucosal tissue.

Topical formulations comprising one or more sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used as preventive, e.g., chemopreventive, compositions. When used in a chemopreventive method, susceptible skin is treated prior to any visible condition in a particular individual.

Sirtuin-modulating compounds may be delivered locally or systemically to a subject. In one embodiment, a sirtuin-modulating compound is delivered locally to a tissue or organ of a subject by injection, topical formulation, etc.

In another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be used for treating or preventing a disease or condition induced or exacerbated by cellular senescence in a subject; methods for decreasing the rate of senescence of a subject, e.g., after onset of senescence; methods for extending the lifespan of a subject; methods for treating or preventing a disease or condition relating to lifespan; methods for treating or preventing a disease or condition relating to the proliferative capacity of cells; and methods for treating or preventing a disease or condition resulting from cell damage or death. In certain embodiments, the method does not act by decreasing the rate of occurrence of diseases that shorten the lifespan of a subject. In certain embodiments, a method does not act by reducing the lethality caused by a disease, such as cancer.

In yet another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered to a subject in order to generally increase the lifespan of its cells and to protect its cells against stress and/or against apoptosis. It is believed that treating a subject with a compound described herein is similar to subjecting the subject to hormesis, i.e., mild stress that is beneficial to organisms and may extend their lifespan.

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered to a subject to prevent aging and aging-related consequences or diseases, such as stroke, heart disease, heart failure, arthritis, high blood pressure, and Alzheimer's disease. Other conditions that can be treated include ocular disorders, e.g., associated with the aging of the eye, such as cataracts, glaucoma, and macular degeneration. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can also be administered to subjects for treatment of diseases, e.g., chronic diseases, associated with cell death, in order to protect the cells from cell death. Exemplary diseases include those associated with neural cell death, neuronal dysfunction, or muscular cell death or dysfunction, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and muscular dystrophy; AIDS; fulminant hepatitis; diseases linked to degeneration of the brain, such as Creutzfeld-Jakob disease, retinitis pigmentosa and cerebellar degeneration; myelodysplasia such as aplastic anemia; ischemic diseases such as myocardial infarction and stroke; hepatic diseases such as alcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such as osteoarthritis; atherosclerosis; alopecia; damage to the skin due to UV light; lichen planus; atrophy of the skin; cataract; and graft rejections. Cell death can also be caused by surgery, drug therapy, chemical exposure or radiation exposure.

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can also be administered to a subject suffering from an acute disease, e.g., damage to an organ or tissue, e.g., a subject suffering from stroke or myocardial infarction or a subject suffering from a spinal cord injury. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used to repair an alcoholic's liver.

Cardiovascular Disease

In another embodiment, the invention provides a method for treating and/or preventing a cardiovascular disease by administering to a subject in need thereof a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein.

Cardiovascular diseases that can be treated or prevented using the sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein include cardiomyopathy or myocarditis; such as idiopathic cardiomyopathy, metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic cardiomyopathy, and hypertensive cardiomyopathy. Also treatable or preventable using compounds and methods described herein are atheromatous disorders of the major blood vessels (macrovascular disease) such as the aorta, the coronary arteries, the carotid arteries, the cerebrovascular arteries, the renal arteries, the iliac arteries, the femoral arteries, and the popliteal arteries. Other vascular diseases that can be treated or prevented include those related to platelet aggregation, the retinal arterioles, the glomerular arterioles, the vasa nervorum, cardiac arterioles, and associated capillary beds of the eye, the kidney, the heart, and the central and peripheral nervous systems. The sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used for increasing HDL levels in plasma of an individual.

Yet other disorders that may be treated with sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein include restenosis, e.g., following coronary intervention, and disorders relating to an abnormal level of high density and low density cholesterol.

In one embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered as part of a combination therapeutic with another cardiovascular agent. In one embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered as part of a combination therapeutic with an anti-arrhythmia agent. In another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered as part of a combination therapeutic with another cardiovascular agent.

Cell Death/Cancer

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered to subjects who have recently received or are likely to receive a dose of radiation or toxin. In one embodiment, the dose of radiation or toxin is received as part of a work-related or medical procedure, e.g., administered as a prophylactic measure. In another embodiment, the radiation or toxin exposure is received unintentionally. In such a case, the compound is preferably administered as soon as possible after the exposure to inhibit apoptosis and the subsequent development of acute radiation syndrome.

Sirtuin-modulating compounds may also be used for treating and/or preventing cancer. In certain embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for treating and/or preventing cancer. Calorie restriction has been linked to a reduction in the incidence of age-related disorders including cancer. Accordingly, an increase in the level and/or activity of a sirtuin protein may be useful for treating and/or preventing the incidence of age-related disorders, such as, for example, cancer. Exemplary cancers that may be treated using a sirtuin-modulating compound are those of the brain and kidney; hormone-dependent cancers including breast, prostate, testicular, and ovarian cancers; lymphomas, and leukemias. In cancers associated with solid tumors, a modulating compound may be administered directly into the tumor. Cancer of blood cells, e.g., leukemia, can be treated by administering a modulating compound into the blood stream or into the bone marrow. Benign cell growth, e.g., warts, can also be treated. Other diseases that can be treated include autoimmune diseases, e.g., systemic lupus erythematosus, scleroderma, and arthritis, in which autoimmune cells should be removed. Viral infections such as herpes, HIV, adenovirus, and HTLV-1 associated malignant and benign disorders can also be treated by administration of sirtuin-modulating compound. Alternatively, cells can be obtained from a subject, treated ex vivo to remove certain undesirable cells, e.g., cancer cells, and administered back to the same or a different subject.

Chemotherapeutic agents may be co-administered with modulating compounds described herein as having anti-cancer activity, e.g., compounds that induce apoptosis, compounds that reduce lifespan or compounds that render cells sensitive to stress. Chemotherapeutic agents may be used by themselves with a sirtuin-modulating compound described herein as inducing cell death or reducing lifespan or increasing sensitivity to stress and/or in combination with other chemotherapeutics agents. In addition to conventional chemotherapeutics, the sirtuin-modulating compounds described herein may also be used with antisense RNA, RNAi or other polynucleotides to inhibit the expression of the cellular components that contribute to unwanted cellular proliferation.

Combination therapies comprising sirtuin-modulating compounds and a conventional chemotherapeutic agent may be advantageous over combination therapies known in the art because the combination allows the conventional chemotherapeutic agent to exert greater effect at lower dosage. In a preferred embodiment, the effective dose (ED₅₀) for a chemotherapeutic agent, or combination of conventional chemotherapeutic agents, when used in combination with a sirtuin-modulating compound is at least 2 fold less than the ED₅₀ for the chemotherapeutic agent alone, and even more preferably at 5 fold, 10 fold or even 25 fold less. Conversely, the therapeutic index (TI) for such chemotherapeutic agent or combination of such chemotherapeutic agent when used in combination with a sirtuin-modulating compound described herein can be at least 2 fold greater than the TI for conventional chemotherapeutic regimen alone, and even more preferably at 5 fold, 10 fold or even 25 fold greater.

Neuronal Diseases/Disorders

In certain aspects, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used to treat patients suffering from neurodegenerative diseases, and traumatic or mechanical injury to the central nervous system (CNS), spinal cord or peripheral nervous system (PNS). Neurodegenerative disease typically involves reductions in the mass and volume of the human brain, which may be due to the atrophy and/or death of brain cells, which are far more profound than those in a healthy person that are attributable to aging. Neurodegenerative diseases can evolve gradually, after a long period of normal brain function, due to progressive degeneration (e.g., nerve cell dysfunction and death) of specific brain regions. Alternatively, neurodegenerative diseases can have a quick onset, such as those associated with trauma or toxins. The actual onset of brain degeneration may precede clinical expression by many years. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), diffuse Lewy body disease, chorea-acanthocytosis, primary lateral sclerosis, ocular diseases (ocular neuritis), chemotherapy-induced neuropathies (e.g., from vincristine, paclitaxel, bortezomib), diabetes-induced neuropathies and Friedreich's ataxia. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used to treat these disorders and others as described below.

AD is a CNS disorder that results in memory loss, unusual behavior, personality changes, and a decline in thinking abilities. These losses are related to the death of specific types of brain cells and the breakdown of connections and their supporting network (e.g. glial cells) between them. The earliest symptoms include loss of recent memory, faulty judgment, and changes in personality. PD is a CNS disorder that results in uncontrolled body movements, rigidity, tremor, and dyskinesia, and is associated with the death of brain cells in an area of the brain that produces dopamine. ALS (motor neuron disease) is a CNS disorder that attacks the motor neurons, components of the CNS that connect the brain to the skeletal muscles.

HD is another neurodegenerative disease that causes uncontrolled movements, loss of intellectual faculties, and emotional disturbance. Tay-Sachs disease and Sandhoff disease are glycolipid storage diseases where GM2 ganglioside and related glycolipids substrates for β-hexosaminidase accumulate in the nervous system and trigger acute neurodegeneration.

It is well-known that apoptosis plays a role in AIDS pathogenesis in the immune system. However, HIV-1 also induces neurological disease, which can be treated with sirtuin-modulating compounds of the invention.

Neuronal loss is also a salient feature of prion diseases, such as Creutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease), Scrapie Disease in sheep and goats, and feline spongiform encephalopathy (FSE) in cats. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be useful for treating or preventing neuronal loss due to these prior diseases.

In another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be used to treat or prevent any disease or disorder involving axonopathy. Distal axonopathy is a type of peripheral neuropathy that results from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of nerves to metabolic or toxic disturbances, and as such may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. Those with distal axonopathies usually present with symmetrical glove-stocking sensori-motor disturbances. Deep tendon reflexes and autonomic nervous system (ANS) functions are also lost or diminished in affected areas.

Diabetic neuropathies are neuropathic disorders that are associated with diabetes mellitus. Relatively common conditions which may be associated with diabetic neuropathy include third nerve palsy; mononeuropathy; mononeuritis multiplex; diabetic amyotrophy; a painful polyneuropathy; autonomic neuropathy; and thoracoabdominal neuropathy.

Peripheral neuropathy is the medical term for damage to nerves of the peripheral nervous system, which may be caused either by diseases of the nerve or from the side-effects of systemic illness. Major causes of peripheral neuropathy include seizures, nutritional deficiencies, and HIV, though diabetes is the most likely cause.

In an exemplary embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be used to treat or prevent multiple sclerosis (MS), including relapsing MS and monosymptomatic MS, and other demyelinating conditions, such as, for example, chromic inflammatory demyelinating polyneuropathy (CIDP), or symptoms associated therewith.

In yet another embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be used to treat trauma to the nerves, including, trauma due to disease, injury (including surgical intervention), or environmental trauma (e.g., neurotoxins, alcoholism, etc.).

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be useful to prevent, treat, and alleviate symptoms of various PNS disorders. The term “peripheral neuropathy” encompasses a wide range of disorders in which the nerves outside of the brain and spinal cord—peripheral nerves—have been damaged. Peripheral neuropathy may also be referred to as peripheral neuritis, or if many nerves are involved, the terms polyneuropathy or polyneuritis may be used.

PNS diseases treatable with sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein include: diabetes, leprosy, Charcot-Marie-Tooth disease, Guillain-Barré syndrome and Brachial Plexus Neuropathies (diseases of the cervical and first thoracic roots, nerve trunks, cords, and peripheral nerve components of the brachial plexus.

In another embodiment, a sirtuin activating compound may be used to treat or prevent a polyglutamine disease. Exemplary polyglutamine diseases include Spinobulbar muscular atrophy (Kennedy disease), Huntington's Disease (HD), Dentatorubral-pallidoluysian atrophy (Haw River syndrome), Spinocerebellar ataxia type 1, Spinocerebellar ataxia type 2, Spinocerebellar ataxia type 3 (Machado-Joseph disease), Spinocerebellar ataxia type 6, Spinocerebellar ataxia type 7, and Spinocerebellar ataxia type 17.

In certain embodiments, the invention provides a method to treat a central nervous system cell to prevent damage in response to a decrease in blood flow to the cell. Typically the severity of damage that may be prevented will depend in large part on the degree of reduction in blood flow to the cell and the duration of the reduction. In one embodiment, apoptotic or necrotic cell death may be prevented. In still a further embodiment, ischemic-mediated damage, such as cytoxic edema or central nervous system tissue anoxemia, may be prevented. In each embodiment, the central nervous system cell may be a spinal cell or a brain cell.

Another aspect encompasses administrating a sirtuin activating compound to a subject to treat a central nervous system ischemic condition. A number of central nervous system ischemic conditions may be treated by the sirtuin activating compounds described herein. In one embodiment, the ischemic condition is a stroke that results in any type of ischemic central nervous system damage, such as apoptotic or necrotic cell death, cytoxic edema or central nervous system tissue anoxia. The stroke may impact any area of the brain or be caused by any etiology commonly known to result in the occurrence of a stroke. In one alternative of this embodiment, the stroke is a brain stem stroke. In another alternative of this embodiment, the stroke is a cerebellar stroke. In still another embodiment, the stroke is an embolic stroke. In yet another alternative, the stroke may be a hemorrhagic stroke. In a further embodiment, the stroke is a thrombotic stroke.

In yet another aspect, a sirtuin activating compound may be administered to reduce infarct size of the ischemic core following a central nervous system ischemic condition. Moreover, a sirtuin activating compound may also be beneficially administered to reduce the size of the ischemic penumbra or transitional zone following a central nervous system ischemic condition.

In one embodiment, a combination drug regimen may include drugs or compounds for the treatment or prevention of neurodegenerative disorders or secondary conditions associated with these conditions. Thus, a combination drug regimen may include one or more sirtuin activators and one or more anti-neurodegeneration agents.

Blood Coagulation Disorders

In other aspects, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used to treat or prevent blood coagulation disorders (or hemostatic disorders). As used interchangeably herein, the terms “hemostasis”, “blood coagulation,” and “blood clotting” refer to the control of bleeding, including the physiological properties of vasoconstriction and coagulation. Blood coagulation assists in maintaining the integrity of mammalian circulation after injury, inflammation, disease, congenital defect, dysfunction or other disruption. Further, the formation of blood clots does not only limit bleeding in case of an injury (hemostasis), but may lead to serious organ damage and death in the context of atherosclerotic diseases by occlusion of an important artery or vein. Thrombosis is thus blood clot formation at the wrong time and place.

Accordingly, the present invention provides anticoagulation and antithrombotic treatments aiming at inhibiting the formation of blood clots in order to prevent or treat blood coagulation disorders, such as myocardial infarction, stroke, loss of a limb by peripheral artery disease or pulmonary embolism.

As used interchangeably herein, “modulating or modulation of hemostasis” and “regulating or regulation of hemostasis” includes the induction (e.g., stimulation or increase) of hemostasis, as well as the inhibition (e.g., reduction or decrease) of hemostasis.

In one aspect, the invention provides a method for reducing or inhibiting hemostasis in a subject by administering a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. The compositions and methods disclosed herein are useful for the treatment or prevention of thrombotic disorders. As used herein, the term “thrombotic disorder” includes any disorder or condition characterized by excessive or unwanted coagulation or hemostatic activity, or a hypercoagulable state. Thrombotic disorders include diseases or disorders involving platelet adhesion and thrombus formation, and may manifest as an increased propensity to form thromboses, e.g., an increased number of thromboses, thrombosis at an early age, a familial tendency towards thrombosis, and thrombosis at unusual sites.

In another embodiment, a combination drug regimen may include drugs or compounds for the treatment or prevention of blood coagulation disorders or secondary conditions associated with these conditions. Thus, a combination drug regimen may include one or more sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein and one or more anti-coagulation or anti-thrombosis agents.

Weight Control

In another aspect, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for treating or preventing weight gain or obesity in a subject. For example, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used, for example, to treat or prevent hereditary obesity, dietary obesity, hormone related obesity, obesity related to the administration of medication, to reduce the weight of a subject, or to reduce or prevent weight gain in a subject. A subject in need of such a treatment may be a subject who is obese, likely to become obese, overweight, or likely to become overweight. Subjects who are likely to become obese or overweight can be identified, for example, based on family history, genetics, diet, activity level, medication intake, or various combinations thereof.

In yet other embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered to subjects suffering from a variety of other diseases and conditions that may be treated or prevented by promoting weight loss in the subject. Such diseases include, for example, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, type 2 diabetes, insulin resistance, glucose intolerance, hyperinsulinemia, coronary heart disease, angina pectoris, congestive heart failure, stroke, gallstones, cholecystitis and cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and respiratory problems, some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation), bladder control problems (such as stress incontinence); uric acid nephrolithiasis; psychological disorders (such as depression, eating disorders, distorted body image, and low self esteem). Finally, patients with AIDS can develop lipodystrophy or insulin resistance in response to combination therapies for AIDS.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for inhibiting adipogenesis or fat cell differentiation, whether in vitro or in vivo. Such methods may be used for treating or preventing obesity.

In other embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for reducing appetite and/or increasing satiety, thereby causing weight loss or avoidance of weight gain. A subject in need of such a treatment may be a subject who is overweight, obese or a subject likely to become overweight or obese. The method may comprise administering daily or, every other day, or once a week, a dose, e.g., in the form of a pill, to a subject. The dose may be an “appetite reducing dose.”

In an exemplary embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered as a combination therapy for treating or preventing weight gain or obesity. For example, one or more sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered in combination with one or more anti-obesity agents.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered to reduce drug-induced weight gain. For example, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be administered as a combination therapy with medications that may stimulate appetite or cause weight gain, in particular, weight gain due to factors other than water retention.

Metabolic Disorders/Diabetes

In another aspect, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for treating or preventing a metabolic disorder, such as insulin-resistance, a pre-diabetic state, type II diabetes, and/or complications thereof. Administration of a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may increase insulin sensitivity and/or decrease insulin levels in a subject. A subject in need of such a treatment may be a subject who has insulin resistance or other precursor symptom of type II diabetes, who has type II diabetes, or who is likely to develop any of these conditions. For example, the subject may be a subject having insulin resistance, e.g., having high circulating levels of insulin and/or associated conditions, such as hyperlipidemia, dyslipogenesis, hypercholesterolemia, impaired glucose tolerance, high blood glucose sugar level, other manifestations of syndrome X, hypertension, atherosclerosis and lipodystrophy.

In an exemplary embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered as a combination therapy for treating or preventing a metabolic disorder. For example, one or more sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered in combination with one or more anti-diabetic agents.

Inflammatory Diseases

In other aspects, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used to treat or prevent a disease or disorder associated with inflammation. Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered prior to the onset of, at, or after the initiation of inflammation. When used prophylactically, the compounds are preferably provided in advance of any inflammatory response or symptom. Administration of the compounds may prevent or attenuate inflammatory responses or symptoms.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to treat or prevent allergies and respiratory conditions, including asthma, bronchitis, pulmonary fibrosis, allergic rhinitis, oxygen toxicity, emphysema, chronic bronchitis, acute respiratory distress syndrome, and any chronic obstructive pulmonary disease (COPD). The compounds may be used to treat chronic hepatitis infection, including hepatitis B and hepatitis C.

Additionally, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to treat autoimmune diseases, and/or inflammation associated with autoimmune diseases, such as arthritis, including rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis, as well as organ-tissue autoimmune diseases (e.g., Raynaud's syndrome), ulcerative colitis, Crohn's disease, oral mucositis, scleroderma, myasthenia gravis, transplant rejection, endotoxin shock, sepsis, psoriasis, eczema, dermatitis, multiple sclerosis, autoimmune thyroiditis, uveitis, systemic lupus erythematosis, Addison's disease, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), and Grave's disease.

In certain embodiments, one or more sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be taken alone or in combination with other compounds useful for treating or preventing inflammation.

Flushing

In another aspect, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for reducing the incidence or severity of flushing and/or hot flashes which are symptoms of a disorder. For instance, the subject method includes the use of sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein, alone or in combination with other agents, for reducing incidence or severity of flushing and/or hot flashes in cancer patients. In other embodiments, the method provides for the use of sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein to reduce the incidence or severity of flushing and/or hot flashes in menopausal and post-menopausal woman.

In another aspect, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used as a therapy for reducing the incidence or severity of flushing and/or hot flashes which are side-effects of another drug therapy, e.g., drug-induced flushing. In certain embodiments, a method for treating and/or preventing drug-induced flushing comprises administering to a patient in need thereof a formulation comprising at least one flushing inducing compound and at least one sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein. In other embodiments, a method for treating drug induced flushing comprises separately administering one or more compounds that induce flushing and one or more sirtuin-modulating compounds, e.g., wherein the sirtuin-modulating compound and flushing inducing agent have not been formulated in the same compositions. When using separate formulations, the sirtuin-modulating compound may be administered (1) at the same as administration of the flushing inducing agent, (2) intermittently with the flushing inducing agent, (3) staggered relative to administration of the flushing inducing agent, (4) prior to administration of the flushing inducing agent, (5) subsequent to administration of the flushing inducing agent, and (6) various combination thereof. Exemplary flushing inducing agents include, for example, niacin, raloxifene, antidepressants, anti-psychotics, chemotherapeutics, calcium channel blockers, and antibiotics.

In one embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to reduce flushing side effects of a vasodilator or an antilipemic agent (including anticholesteremic agents and lipotropic agents). In an exemplary embodiment, a sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be used to reduce flushing associated with the administration of niacin.

In another embodiment, the invention provides a method for treating and/or preventing hyperlipidemia with reduced flushing side effects. In another representative embodiment, the method involves the use of sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein to reduce flushing side effects of raloxifene. In another representative embodiment, the method involves the use of sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein to reduce flushing side effects of antidepressants or anti-psychotic agent. For instance, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used in conjunction (administered separately or together) with a serotonin reuptake inhibitor, or a 5HT2 receptor antagonist.

In certain embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used as part of a treatment with a serotonin reuptake inhibitor (SRI) to reduce flushing. In still another representative embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to reduce flushing side effects of chemotherapeutic agents, such as cyclophosphamide and tamoxifen.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to reduce flushing side effects of calcium channel blockers, such as amlodipine.

In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used to reduce flushing side effects of antibiotics. For example, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be used in combination with levofloxacin.

Ocular Disorders

One aspect of the present invention is a method for inhibiting, reducing or otherwise treating vision impairment by administering to a patient a therapeutic dosage of sirtuin modulator selected from a compound disclosed herein, or a pharmaceutically acceptable salt, prodrug or a metabolic derivative thereof.

In certain aspects of the invention, the vision impairment is caused by damage to the optic nerve or central nervous system. In particular embodiments, optic nerve damage is caused by high intraocular pressure, such as that created by glaucoma. In other particular embodiments, optic nerve damage is caused by swelling of the nerve, which is often associated with an infection or an immune (e.g., autoimmune) response such as in optic neuritis.

In certain aspects of the invention, the vision impairment is caused by retinal damage. In particular embodiments, retinal damage is caused by disturbances in blood flow to the eye (e.g., arteriosclerosis, vasculitis). In particular embodiments, retinal damage is caused by disruption of the macula (e.g., exudative or non-exudative macular degeneration).

Exemplary retinal diseases include Exudative Age Related Macular Degeneration, Nonexudative Age Related Macular Degeneration, Retinal Electronic Prosthesis and RPE Transplantation Age Related Macular Degeneration, Acute Multifocal Placoid Pigment Epitheliopathy, Acute Retinal Necrosis, Best Disease, Branch Retinal Artery Occlusion, Branch Retinal Vein Occlusion, Cancer Associated and Related Autoimmune Retinopathies, Central Retinal Artery Occlusion, Central Retinal Vein Occlusion, Central Serous Chorioretinopathy, Eales Disease, Epimacular Membrane, Lattice Degeneration, Macroaneurysm, Diabetic Macular Edema, Irvine-Gass Macular Edema, Macular Hole, Subretinal Neovascular Membranes, Diffuse Unilateral Subacute Neuroretinitis, Nonpseudophakic Cystoid Macular Edema, Presumed Ocular Histoplasmosis Syndrome, Exudative Retinal Detachment, Postoperative Retinal Detachment, Proliferative Retinal Detachment, Rhegmatogenous Retinal Detachment, Tractional Retinal Detachment, Retinitis Pigmentosa, CMV Retinitis, Retinoblastoma, Retinopathy of Prematurity, Birdshot Retinopathy, Background Diabetic Retinopathy, Proliferative Diabetic Retinopathy, Hemoglobinopathies Retinopathy, Purtscher Retinopathy, Valsalva Retinopathy, Juvenile Retinoschisis, Senile Retinoschisis, Terson Syndrome and White Dot Syndromes.

Other exemplary diseases include ocular bacterial infections (e.g. conjunctivitis, keratitis, tuberculosis, syphilis, gonorrhea), viral infections (e.g. Ocular Herpes Simplex Virus, Varicella Zoster Virus, Cytomegalovirus retinitis, Human Immunodeficiency Virus (HIV)) as well as progressive outer retinal necrosis secondary to HIV or other HIV-associated and other immunodeficiency-associated ocular diseases. In addition, ocular diseases include fungal infections (e.g. Candida choroiditis, histoplasmosis), protozoal infections (e.g. toxoplasmosis) and others such as ocular toxocariasis and sarcoidosis.

One aspect of the invention is a method for inhibiting, reducing or treating vision impairment in a subject undergoing treatment with a chemotherapeutic drug (e.g., a neurotoxic drug, a drug that raises intraocular pressure such as a steroid), by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin modulator disclosed herein.

Another aspect of the invention is a method for inhibiting, reducing or treating vision impairment in a subject undergoing surgery, including ocular or other surgeries performed in the prone position such as spinal cord surgery, by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin modulator disclosed herein. Ocular surgeries include cataract, iridotomy and lens replacements.

Another aspect of the invention is the treatment, including inhibition and prophylactic treatment, of age related ocular diseases include cataracts, dry eye, age-related macular degeneration (AMD), retinal damage and the like, by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin modulator disclosed herein.

Another aspect of the invention is the prevention or treatment of damage to the eye caused by stress, chemical insult or radiation, by administering to the subject in need of such treatment a therapeutic dosage of a sirtuin modulator disclosed herein. Radiation or electromagnetic damage to the eye can include that caused by CRT's or exposure to sunlight or UV.

In one embodiment, a combination drug regimen may include drugs or compounds for the treatment or prevention of ocular disorders or secondary conditions associated with these conditions. Thus, a combination drug regimen may include one or more sirtuin activators and one or more therapeutic agents for the treatment of an ocular disorder.

In one embodiment, a sirtuin modulator can be administered in conjunction with a therapy for reducing intraocular pressure. In another embodiment, a sirtuin modulator can be administered in conjunction with a therapy for treating and/or preventing glaucoma. In yet another embodiment, a sirtuin modulator can be administered in conjunction with a therapy for treating and/or preventing optic neuritis. In one embodiment, a sirtuin modulator can be administered in conjunction with a therapy for treating and/or preventing CMV Retinopathy. In another embodiment, a sirtuin modulator can be administered in conjunction with a therapy for treating and/or preventing multiple sclerosis.

Mitochondrial-Associated Diseases and Disorders

In certain embodiments, the invention provides methods for treating diseases or disorders that would benefit from increased mitochondrial activity. The methods involve administering to a subject in need thereof a therapeutically effective amount of a sirtuin activating compound. Increased mitochondrial activity refers to increasing activity of the mitochondria while maintaining the overall numbers of mitochondria (e.g., mitochondrial mass), increasing the numbers of mitochondria thereby increasing mitochondrial activity (e.g., by stimulating mitochondrial biogenesis), or combinations thereof. In certain embodiments, diseases and disorders that would benefit from increased mitochondrial activity include diseases or disorders associated with mitochondrial dysfunction.

In certain embodiments, methods for treating diseases or disorders that would benefit from increased mitochondrial activity may comprise identifying a subject suffering from a mitochondrial dysfunction. Methods for diagnosing a mitochondrial dysfunction may involve molecular genetic, pathologic and/or biochemical analyses. Diseases and disorders associated with mitochondrial dysfunction include diseases and disorders in which deficits in mitochondrial respiratory chain activity contribute to the development of pathophysiology of such diseases or disorders in a mammal. Diseases or disorders that would benefit from increased mitochondrial activity generally include for example, diseases in which free radical mediated oxidative injury leads to tissue degeneration, diseases in which cells inappropriately undergo apoptosis, and diseases in which cells fail to undergo apoptosis.

In certain embodiments, the invention provides methods for treating a disease or disorder that would benefit from increased mitochondrial activity that involves administering to a subject in need thereof one or more sirtuin activating compounds in combination with another therapeutic agent such as, for example, an agent useful for treating mitochondrial dysfunction or an agent useful for reducing a symptom associated with a disease or disorder involving mitochondrial dysfunction.

In exemplary embodiments, the invention provides methods for treating diseases or disorders that would benefit from increased mitochondrial activity by administering to a subject a therapeutically effective amount of a sirtuin activating compound. Exemplary diseases or disorders include, for example, neuromuscular disorders (e.g., Friedreich's Ataxia, muscular dystrophy, multiple sclerosis, etc.), disorders of neuronal instability (e.g., seizure disorders, migraine, etc.), developmental delay, neurodegenerative disorders (e.g., Alzheimer's Disease, Parkinson's Disease, amyotrophic lateral sclerosis, etc.), ischemia, renal tubular acidosis, age-related neurodegeneration and cognitive decline, chemotherapy fatigue, age-related or chemotherapy-induced menopause or irregularities of menstrual cycling or ovulation, mitochondrial myopathies, mitochondrial damage (e.g., calcium accumulation, excitotoxicity, nitric oxide exposure, hypoxia, etc.), and mitochondrial deregulation.

Muscular dystrophy refers to a family of diseases involving deterioration of neuromuscular structure and function, often resulting in atrophy of skeletal muscle and myocardial dysfunction, such as Duchenne muscular dystrophy. In certain embodiments, sirtuin activating compounds may be used for reducing the rate of decline in muscular functional capacities and for improving muscular functional status in patients with muscular dystrophy.

In certain embodiments, sirtuin modulating compounds may be useful for treatment mitochondrial myopathies. Mitochondrial myopathies range from mild, slowly progressive weakness of the extraocular muscles to severe, fatal infantile myopathies and multisystem encephalomyopathies. Some syndromes have been defined, with some overlap between them. Established syndromes affecting muscle include progressive external ophthalmoplegia, the Kearns-Sayre syndrome (with ophthalmoplegia, pigmentary retinopathy, cardiac conduction defects, cerebellar ataxia, and sensorineural deafness), the MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), the MERFF syndrome (myoclonic epilepsy and ragged red fibers), limb-girdle distribution weakness, and infantile myopathy (benign or severe and fatal).

In certain embodiments, sirtuin activating compounds may be useful for treating patients suffering from toxic damage to mitochondria, such as, toxic damage due to calcium accumulation, excitotoxicity, nitric oxide exposure, drug induced toxic damage, or hypoxia.

In certain embodiments, sirtuin activating compounds may be useful for treating diseases or disorders associated with mitochondrial deregulation.

Muscle Performance

In other embodiments, the invention provides methods for enhancing muscle performance by administering a therapeutically effective amount of a sirtuin activating compound. For example, sirtuin activating compounds may be useful for improving physical endurance (e.g., ability to perform a physical task such as exercise, physical labor, sports activities, etc), inhibiting or retarding physical fatigues, enhancing blood oxygen levels, enhancing energy in healthy individuals, enhance working capacity and endurance, reducing muscle fatigue, reducing stress, enhancing cardiac and cardiovascular function, improving sexual ability, increasing muscle ATP levels, and/or reducing lactic acid in blood. In certain embodiments, the methods involve administering an amount of a sirtuin activating compound that increase mitochondrial activity, increase mitochondrial biogenesis, and/or increase mitochondrial mass.

Sports performance refers to the ability of the athlete's muscles to perform when participating in sports activities. Enhanced sports performance, strength, speed and endurance are measured by an increase in muscular contraction strength, increase in amplitude of muscle contraction, shortening of muscle reaction time between stimulation and contraction. Athlete refers to an individual who participates in sports at any level and who seeks to achieve an improved level of strength, speed and endurance in their performance, such as, for example, body builders, bicyclists, long distance runners, short distance runners, etc. Enhanced sports performance in manifested by the ability to overcome muscle fatigue, ability to maintain activity for longer periods of time, and have a more effective workout.

In the arena of athlete muscle performance, it is desirable to create conditions that permit competition or training at higher levels of resistance for a prolonged period of time.

It is contemplated that the methods of the present invention will also be effective in the treatment of muscle related pathological conditions, including acute sarcopenia, for example, muscle atrophy and/or cachexia associated with burns, bed rest, limb immobilization, or major thoracic, abdominal, and/or orthopedic surgery.

In certain embodiments, the invention provides novel dietary compositions comprising sirtuin modulators, a method for their preparation, and a method of using the compositions for improvement of sports performance. Accordingly, provided are therapeutic compositions, foods and beverages that have actions of improving physical endurance and/or inhibiting physical fatigues for those people involved in broadly-defined exercises including sports requiring endurance and labors requiring repeated muscle exertions. Such dietary compositions may additional comprise electrolytes, caffeine, vitamins, carbohydrates, etc.

Other Uses

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for treating or preventing viral infections (such as infections by influenza, herpes or papilloma virus) or as antifungal agents. In certain embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered as part of a combination drug therapy with another therapeutic agent for the treatment of viral diseases. In another embodiment, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be administered as part of a combination drug therapy with another anti-fungal agent.

Subjects that may be treated as described herein include eukaryotes, such as mammals, e.g., humans, ovines, bovines, equines, porcines, canines, felines, non-human primate, mice, and rats. Cells that may be treated include eukaryotic cells, e.g., from a subject described above, or plant cells, yeast cells and prokaryotic cells, e.g., bacterial cells. For example, modulating compounds may be administered to farm animals to improve their ability to withstand farming conditions longer.

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used to increase lifespan, stress resistance, and resistance to apoptosis in plants. In one embodiment, a compound is applied to plants, e.g., on a periodic basis, or to fungi. In another embodiment, plants are genetically modified to produce a compound. In another embodiment, plants and fruits are treated with a compound prior to picking and shipping to increase resistance to damage during shipping. Plant seeds may also be contacted with compounds described herein, e.g., to preserve them.

In other embodiments, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may be used for modulating lifespan in yeast cells. Situations in which it may be desirable to extend the lifespan of yeast cells include any process in which yeast is used, e.g., the making of beer, yogurt, and bakery items, e.g., bread. Use of yeast having an extended lifespan can result in using less yeast or in having the yeast be active for longer periods of time. Yeast or other mammalian cells used for recombinantly producing proteins may also be treated as described herein.

Sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used to increase lifespan, stress resistance and resistance to apoptosis in insects. In this embodiment, compounds would be applied to useful insects, e.g., bees and other insects that are involved in pollination of plants. In a specific embodiment, a compound would be applied to bees involved in the production of honey. Generally, the methods described herein may be applied to any organism, e.g., eukaryote, which may have commercial importance. For example, they can be applied to fish (aquaculture) and birds (e.g., chicken and fowl).

Higher doses of sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein may also be used as a pesticide by interfering with the regulation of silenced genes and the regulation of apoptosis during development. In this embodiment, a compound may be applied to plants using a method known in the art that ensures the compound is bio-available to insect larvae, and not to plants.

At least in view of the link between reproduction and longevity, sirtuin-modulating compounds that increase the level and/or activity of a sirtuin protein can be applied to affect the reproduction of organisms such as insects, animals and microorganisms.

4. Assays

Yet other methods contemplated herein include screening methods for identifying compounds or agents that modulate sirtuins. An agent may be a nucleic acid, such as an aptamer. Assays may be conducted in a cell based or cell free format. For example, an assay may comprise incubating (or contacting) a sirtuin with a test agent under conditions in which a sirtuin can be modulated by an agent known to modulate the sirtuin, and monitoring or determining the level of modulation of the sirtuin in the presence of the test agent relative to the absence of the test agent. The level of modulation of a sirtuin can be determined by determining its ability to deacetylate a substrate. Exemplary substrates are acetylated peptides which can be obtained from BIOMOL (Plymouth Meeting, Pa.). Preferred substrates include peptides of p53, such as those comprising an acetylated K382. A particularly preferred substrate is the Fluor de Lys-SIRT1 (BIOMOL), i.e., the acetylated peptide Arg-His-Lys-Lys. Other substrates are peptides from human histones H3 and H4 or an acetylated amino acid. Substrates may be fluorogenic. The sirtuin may be SIRT1, Sir2, SIRT3, or a portion thereof. For example, recombinant SIRT1 can be obtained from BIOMOL. The reaction may be conducted for about 30 minutes and stopped, e.g., with nicotinamide. The HDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOL Research Laboratories) may be used to determine the level of acetylation. Similar assays are described in Bitterman et al. (2002) J. Biol. Chem. 277:45099. The level of modulation of the sirtuin in an assay may be compared to the level of modulation of the sirtuin in the presence of one or more (separately or simultaneously) compounds described herein, which may serve as positive or negative controls. Sirtuins for use in the assays may be full length sirtuin proteins or portions thereof. Since it has been shown herein that activating compounds appear to interact with the N-terminus of SIRT1, proteins for use in the assays include N-terminal portions of sirtuins, e.g., about amino acids 1-176 or 1-255 of SIRT1; about amino acids 1-174 or 1-252 of Sir2.

In one embodiment, a screening assay comprises (i) contacting a sirtuin with a test agent and an acetylated substrate under conditions appropriate for the sirtuin to deacetylate the substrate in the absence of the test agent; and (ii) determining the level of acetylation of the substrate, wherein a lower level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent stimulates deacetylation by the sirtuin, whereas a higher level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent inhibits deacetylation by the sirtuin.

Methods for identifying an agent that modulates, e.g., stimulates, sirtuins in vivo may comprise (i) contacting a cell with a test agent and a substrate that is capable of entering a cell in the presence of an inhibitor of class I and class II HDACs under conditions appropriate for the sirtuin to deacetylate the substrate in the absence of the test agent; and (ii) determining the level of acetylation of the substrate, wherein a lower level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent stimulates deacetylation by the sirtuin, whereas a higher level of acetylation of the substrate in the presence of the test agent relative to the absence of the test agent indicates that the test agent inhibits deacetylation by the sirtuin. A preferred substrate is an acetylated peptide, which is also preferably fluorogenic, as further described herein. The method may further comprise lysing the cells to determine the level of acetylation of the substrate. Substrates may be added to cells at a concentration ranging from about 1 μM to about 10 mM, preferably from about 10 μM to 1 mM, even more preferably from about 100 μM to 1 mM, such as about 200 μM. A preferred substrate is an acetylated lysine, e.g., ε-acetyl lysine (Fluor de Lys, FdL) or Fluor de Lys-SIRT1. A preferred inhibitor of class I and class II HDACs is trichostatin A (TSA), which may be used at concentrations ranging from about 0.01 to 100 μM, preferably from about 0.1 to 10 μM, such as 1 μM. Incubation of cells with the test compound and the substrate may be conducted for about 10 minutes to 5 hours, preferably for about 1-3 hours. Since TSA inhibits all class I and class II HDACs, and that certain substrates, e.g., Fluor de Lys, is a poor substrate for SIRT2 and even less a substrate for SIRT3-7, such an assay may be used to identify modulators of SIRT1 in vivo.

5. Pharmaceutical Compositions

The sirtuin-modulating compounds described herein may be formulated in a conventional manner using one or more physiologically or pharmaceutically acceptable carriers or excipients. For example, sirtuin-modulating compounds and their pharmaceutically acceptable salts and solvates may be formulated for administration by, for example, injection (e.g. SubQ, IM, IP), inhalation or insufflation (either through the mouth or the nose) or oral, buccal, sublingual, transdermal, nasal, parenteral or rectal administration. In one embodiment, a sirtuin-modulating compound may be administered locally, at the site where the target cells are present, i.e., in a specific tissue, organ, or fluid (e.g., blood, cerebrospinal fluid, etc.).

Sirtuin-modulating compounds can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For parenteral administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets, lozenges, or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery), sirtuin-modulating compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Sirtuin-modulating compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Sirtuin-modulating compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, sirtuin-modulating compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, sirtuin-modulating compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Controlled release formula also includes patches.

In certain embodiments, the compounds described herein can be formulated for delivery to the central nervous system (CNS) (reviewed in Begley, Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approaches for drug delivery to the CNS include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).

Liposomes are a further drug delivery system which is easily injectable. Accordingly, in the method of invention the active compounds can also be administered in the form of a liposome delivery system. Liposomes are well-known by a person skilled in the art. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine of phosphatidylcholines. Liposomes being usable for the method of invention encompass all types of liposomes including, but not limited to, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.

Another way to produce a formulation, particularly a solution, of a sirtuin modulator such as resveratrol or a derivative thereof, is through the use of cyclodextrin. By cyclodextrin is meant α-, β-, or γ-cyclodextrin. Cyclodextrins are described in detail in Pitha et al., U.S. Pat. No. 4,727,064, which is incorporated herein by reference. Cyclodextrins are cyclic oligomers of glucose; these compounds form inclusion complexes with any drug whose molecule can fit into the lipophile-seeking cavities of the cyclodextrin molecule.

Rapidly disintegrating or dissolving dosage forms are useful for the rapid absorption, particularly buccal and sublingual absorption, of pharmaceutically active agents. Fast melt dosage forms are beneficial to patients, such as aged and pediatric patients, who have difficulty in swallowing typical solid dosage forms, such as caplets and tablets. Additionally, fast melt dosage forms circumvent drawbacks associated with, for example, chewable dosage forms, wherein the length of time an active agent remains in a patient's mouth plays an important role in determining the amount of taste masking and the extent to which a patient may object to throat grittiness of the active agent.

Pharmaceutical compositions (including cosmetic preparations) may comprise from about 0.00001 to 100% such as from 0.001 to 10% or from 0.1% to 5% by weight of one or more sirtuin-modulating compounds described herein. In other embodiments, the pharmaceutical composition comprises: (i) 0.05 to 1000 mg of the compounds of the invention, or a pharmaceutically acceptable salt thereof, and (ii) 0.1 to 2 grams of one or more pharmaceutically acceptable excipients.

In one embodiment, a sirtuin-modulating compound described herein, is incorporated into a topical formulation containing a topical carrier that is generally suited to topical drug administration and comprising any such material known in the art. The topical carrier may be selected so as to provide the composition in the desired form, e.g., as an ointment, lotion, cream, microemulsion, gel, oil, solution, or the like, and may be comprised of a material of either naturally occurring or synthetic origin. It is preferable that the selected carrier not adversely affect the active agent or other components of the topical formulation. Examples of suitable topical carriers for use herein include water, alcohols and other nontoxic organic solvents, glycerin, mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetable oils, parabens, waxes, and the like.

Formulations may be colorless, odorless ointments, lotions, creams, microemulsions and gels.

Sirtuin-modulating compounds may be incorporated into ointments, which generally are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. The specific ointment base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery, and, preferably, will provide for other desired characteristics as well, e.g., emolliency or the like. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.

Sirtuin-modulating compounds may be incorporated into lotions, which generally are preparations to be applied to the skin surface without friction, and are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are usually suspensions of solids, and may comprise a liquid oily emulsion of the oil-in-water type.

Sirtuin-modulating compounds may be incorporated into creams, which generally are viscous liquid or semisolid emulsions, either oil-in-water or water-in-oil. Cream bases are water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation, as explained in Remington's, supra, is generally a nonionic, anionic, cationic or amphoteric surfactant.

Sirtuin-modulating compounds may be incorporated into microemulsions, which generally are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules (Encyclopedia of Pharmaceutical Technology (New York: Marcel Dekker, 1992), volume 9).

Sirtuin-modulating compounds may be incorporated into gel formulations, which generally are semisolid systems consisting of either suspensions made up of small inorganic particles (two-phase systems) or large organic molecules distributed substantially uniformly throughout a carrier liquid (single phase gels). Although gels commonly employ aqueous carrier liquid, alcohols and oils can be used as the carrier liquid as well.

Other active agents may also be included in formulations, e.g., other anti-inflammatory agents, analgesics, antimicrobial agents, antifungal agents, antibiotics, vitamins, antioxidants, and sunblock agents commonly found in sunscreen formulations including, but not limited to, anthranilates, benzophenones (particularly benzophenone-3), camphor derivatives, cinnamates (e.g., octyl methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl methane), p-aminobenzoic acid (PABA) and derivatives thereof, and salicylates (e.g., octyl salicylate).

In certain topical formulations, the active agent is present in an amount in the range of approximately 0.25 wt. % to 75 wt. % of the formulation, preferably in the range of approximately 0.25 wt. % to 30 wt. % of the formulation, more preferably in the range of approximately 0.5 wt. % to 15 wt. % of the formulation, and most preferably in the range of approximately 1.0 wt. % to 10 wt. % of the formulation.

Conditions of the eye can be treated or prevented by, e.g., systemic, topical, intraocular injection of a sirtuin-modulating compound, or by insertion of a sustained release device that releases a sirtuin-modulating compound. A sirtuin-modulating compound that increases the level and/or activity of a sirtuin protein may be delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, as for example the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The pharmaceutically acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil or an encapsulating material. Alternatively, the compounds of the invention may be injected directly into the vitreous and aqueous humour. In a further alternative, the compounds may be administered systemically, such as by intravenous infusion or injection, for treatment of the eye.

Sirtuin-modulating compounds described herein may be stored in oxygen free environment. For example, resveratrol or analog thereof can be prepared in an airtight capsule for oral administration, such as Capsugel from Pfizer, Inc.

Cells, e.g., treated ex vivo with a sirtuin-modulating compound, can be administered according to methods for administering a graft to a subject, which may be accompanied, e.g., by administration of an immunosuppressant drug, e.g., cyclosporin A. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

Toxicity and therapeutic efficacy of sirtuin-modulating compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The LD₅₀ is the dose lethal to 50% of the population. The ED₅₀ is the dose therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects (LD₅₀/ED₅₀) is the therapeutic index. Sirtuin-modulating compounds that exhibit large therapeutic indexes are preferred. While sirtuin-modulating compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may lie within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

6. Kits

Also provided herein are kits, e.g., kits for therapeutic purposes or kits for modulating the lifespan of cells or modulating apoptosis. A kit may comprise one or more sirtuin-modulating compounds, e.g., in premeasured doses. A kit may optionally comprise devices for contacting cells with the compounds and instructions for use. Devices include syringes, stents and other devices for introducing a sirtuin-modulating compound into a subject (e.g., the blood vessel of a subject) or applying it to the skin of a subject.

In yet another embodiment, the invention provides a composition of matter comprising a sirtuin modulator of this invention and another therapeutic agent (the same ones used in combination therapies and combination compositions) in separate dosage forms, but associated with one another. The term “associated with one another” as used herein means that the separate dosage forms are packaged together or otherwise attached to one another such that it is readily apparent that the separate dosage forms are intended to be sold and administered as part of the same regimen. The agent and the sirtuin modulator are preferably packaged together in a blister pack or other multi-chamber package, or as connected, separately sealed containers (such as foil pouches or the like) that can be separated by the user (e.g., by tearing on score lines between the two containers).

In still another embodiment, the invention provides a kit comprising in separate vessels, a) a sirtuin modulator of this invention; and b) another therapeutic agent such as those described elsewhere in the specification.

The practice of the present methods will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention in any way.

Example 1 Synthesis of N-(3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-8-yl)picolinamide (Compound 239) Step 1. Preparation of 8-nitro-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazin-4-one (3)

3-(trifluoromethyl)benzoyl chloride 2 (4.5 mL, 30.2 mmol) was added to a suspension of 2-amino-3-nitrobenzoic acid 1 (5.0 g, 27.5 mmol) in pyridine (65 mL). The reaction mixture was stirred at room temperature for 12 h then poured into ice cooled H₂O (300 mL). The resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 3 (5 g, 51% yield) as a yellow solid which was used without further purification.

Step 2. Preparation of 3-methyl-8-nitro-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (4)

A solution of methylamine (6.3 mL, 12.5 mmol) in THF was added to a suspension of 8-nitro-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazin-4-one 3 (1.5 g, 4.2 mmol) in AcOH (18 mL). The reaction mixture was heated under gentle reflux for 12 h then cooled to room temperature. The volatiles were removed in vacuo and the residue was taken up in EtOAc, washed with sat. aq NaHCO₃, brine, dried (MgSO₄) and concentrated. The residue was purified by MPLC eluting with pentane/EtOAc (0-75%) to give 4 (1.3 g, 89% yield) as a yellow solid.

Step 3. Preparation of 8-amino-3-methyl-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (5)

Pd/C 10 wt % (100 mg) was added to a degassed solution of 3-methyl-8-nitro-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 4 (1.3 g, 3.7 mmol) in THF (35 mL). The mixture was hydrogenated under balloon pressure for 12 h. The catalyst was removed by filtration and the solvent evaporated. The residue was purified by MPLC eluting with pentane/EtOAc (0-50%) to give 5 (1.1 g, 90% yield) as a yellow solid.

Step 4. Preparation of N-(3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-8-yl)picolinamide (Compound 239)

A mixture of 8-amino-3-methyl-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 5 (106 mg, 0.33 mmol), pyridine-2-carboxylic acid 6 (84 mg, 0.68 mmol), HATU (263 mg, 0.69 mmo) and DIPEA (208 mg) in DMF were heated at 55° C. for 2.5 h then poured into H₂O. The resulting precipitate was collected by filtration, rinsed with H₂O, then Et₂O to give Compound 239 as a yellow solid (130 mg, 93% yield). MS (ESI) calc. for C₂₂H₁₅F₃N₄O₂: 424.11. found: 425 [M+H].

Example 2 Synthesis of N-(3-methyl-4-oxo-2-phenyl-3,4-dihydroquinazolin-8-yl)pyridine-3-sulfonamide (Compound 361)

Pyridine-3-sulfonyl chloride hydrochloride 7 (280 mg, 1.3 mmol) was added to a solution of 8-amino-3-methyl-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 5 (100 mg, 0.313 mmol) in pyridine (5 mL). The reaction mixture was heated at 80° C. for 12 h. The pyridine was removed in vacuo. The residue was taken up in CH₂Cl₂, washed with sat. aq NaHCO₃, dried (MgSO₄) and concentrated. The crude residue was purified by MPLC eluting with CH₂Cl₂/MeOH (0-10%) followed by recrystallization from CH₃CN to give Compound 361 (77 mg, 53% yield). MS (ESI) calcd for C₂₁H₁₅F₃N₄O₃S: 460.08. found: 461 [M+H].

Example 3 Synthesis of N-(4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-8-yl)picolinamide (Compound 231) Step 1. Preparation of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (8)

8-nitro-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazin-4-one 3 (150 mg, 0.45 mmol) was added to a solution of ammonia (5.0 mL, 10 mmol) in IPA. The reaction mixture was heated under gentle reflux for 12 h then cooled to room temperature. The reaction mixture was poured into H₂O and the resulting precipitate was collected by filtration rinsed with H₂O and dried under vacuum. The crude residue was recrystallized from EtOAc to give 8 (91 mg, 62% yield) as a yellow solid.

Step 2. Preparation of 8-amino-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one (9)

Compound 9 was prepared by a procedure similar to that reported for 8-amino-3-methyl-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 5 in 99% yield.

Step 3. Preparation of N-(4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-8-yl)picolinamide (Compound 231)

A mixture of 8-amino-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 9 (125 mg, 0.41 mmol), pyrazine-2-carboxylic acid 10 (102 mg, 0.82 mmol), HATU (342 mg, 0.90 mmo) and DIPEA (214 μl, 1.2 mmol) in DMAC (5 mL) were heated at 70° C. for 12 h then poured into H₂O. The precipitate was collected by filtration, rinsed with H₂O then recrystallized from EtOH to give Compound 231 (144 mg, 86% yield) as a white solid. MS (ESI) calc. for C₂₀H₁₂F₃N₅O₂: 411.09. found: 412 [M+H].

Example 4 Synthesis of N-(2-(2,4-dimethylthiazol-5-yl)-3-methyl-4-oxo-3,4-dihydroquinazolin-8-yl)pyrazine-2-carboxamide (Compound 287) Step 1. Preparation of methyl 2-amino-3-nitrobenzoate (11)

2-amino-3-nitrobenzoic acid 1 (5 g, 27.4 mmol) was dissolved in methanol (100 mL) and con H₂SO₄ was added. The mixture was heated at 100° C. for 12 h, concentrated to dryness and suspended in H₂O (50 mL). The mixture was extracted with CH₂Cl₂ (50 mL), dried (MgSO₄) and concentrated. The crude residue was purified by flash chromatography to give 11 (5.1 g, 95% yield).

Step 2. Preparation of methyl 2-(bis-(tert-butoxycarbonyl)amino)-3-nitrobenzoate (12)

(Boc)₂O (5 g, 23 mmol) was added to a solution of methyl 2-amino-3-nitrobenzoate 11 (4.5 g, 23 mmol) and DMAP (5 g, 46 mmol) in THF. The reaction mixture was stirred at room temperature for 6 hours, concentrated and purified by flash chromatography to give 12 (6.4 g, 70% yield).

Step 3. Preparation of methyl 3-amino-2-(bis(tert-butoxycarbonyl)amino)benzoate (13)

Raney Ni (1 g) was added to a solution of methyl 2-(bis-(tert-butoxycarbonyl)amino)-3-nitrobenzoate 12 (6 g, 27.5 mmol) in THF. The reaction mixture was hydrogenated under balloon pressure for 4 h. The catalyst was removed by filtration and the solvent evaporated to give 13 (4.4 g, 80% yield).

Step 4. Preparation of methyl 2-(bis-(tert-butoxycarbonyl)amino)-3-(pyrazine-2-carboxamido)benzoate (14)

A solution of methyl 3-amino-2-(bis(tert-butoxycarbonyl)amino)benzoate 13 (3.8 g, 10.4 mmol), pyrazine-2-carboxylic acid 10 (1.54 g, 12.5 mmol), HATU (7.9 g, 20.8 mmol) and DIPEA (2.68 g, 20.8 mmol) in DMF was heated at 50° C. for 12 h. The reaction mixture was poured into H₂O and the resulting precipitate was collected by filtration and dried under vacuum. The crude residue was purified by flash chromatography to give 14 (2.87 g, 58% yield)

Step 5. Preparation of methyl 2-amino-3-(pyrazine-2-carboxamido)benzoate (15)

HCl gas was bubbled through a solution of methyl 2-(bis-(tert-butoxycarbonyl)amino)-3-(pyrazine-2-carboxamido)benzoate 14 (2.8 g, 5.9 mmol) in MeOH (50 ml) for 2 h. The volatiles were removed in vacuo and H₂O (20 mL) was added. The pH was adjusted to 7 using aq NaOH and the resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 15 (1.37 g, 85% yield).

Step 6. Preparation of 2-amino-3-(pyrazine-2-carboxamido)benzoic acid (16)

A mixture of aq NaOH (20 mL, 88.2 mmol) and methyl 2-amino-3-(pyrazine-2-carboxamido)benzoate 15 (1.2 g, 4.41 mmol) in THF (20 mL) was stirred at room temperature for 4 h. The volatiles were evaporated and the pH adjusted to 4 using aq HCl. The resulting precipitate was collected by filtration, rinsed with H₂O and dried under vacuum to give 16 (750 mg, 66% yield).

Step 7. Preparation of N-(2-(2,4-dimethylthiazol-5-yl)-4-oxo-4H-benzo[d][1,3]oxazin-8-yl)pyrazine-2-carboxamide (18)

2,4-dimethylthiazole-5-carbonyl chloride 17 (104 mg, 0.58 mmol) was added to a suspension of 2-amino-3-(pyrazine-2-carboxamido)benzoic acid 16 (100 mg, 0.39 mmol) in pyridine (5 mL). The reaction mixture was stirred at room temperature for 12 h then poured into ice cooled H₂O (30 mL). The resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 18 (110 mg, 81% yield).

Step 8. Preparation of N-(2-(2,4-dimethylthiazol-5-yl)-3-methyl-4-oxo-3,4-dihydroquinazolin-8-yl)pyrazine-2-carboxamide (Compound 287)

N-(2-(2,4-dimethylthiazol-5-yl)-4-oxo-4H-benzo[d][1,3]oxazin-8-yl)pyrazine-2-carboxamide 18 (100 mg, 0.26 mmol) was dissolved in 30 wt % solution of CH₃NH₂ in methanol (10 mL). The reaction mixture was heated at 80° C. for 12 h. The resulting precipitate was collected by filtration and rinsed with DME to give Compound 287 (32 mg, 31% yield). MS (ESI) calc. for C₁₉H₁₆F₃N₆O₂S: 392.11. found: 393 [M+H].

Example 5 Synthesis of N-(3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazolin-8-yl)-5-oxopyrrolidine-2-carboxamide (Compound 339)

Thionylchloride (83 mg, 0.7 mmol) was added to a solution of 5-oxopyrrolidine-2-carboxylic acid 19 (65 mg, 0.5 mmol) in THF (5 mL) at 0° C. DMF (1 drop) was added and the solution was allowed to warm to room temperature for 3 h then cooled to 0° C. A solution of 8-amino-3-methyl-2-(3-(trifluoromethyl)phenyl)quinazolin-4(3H)-one 5 (65 mg, 0.2 mmol) and triethylamine (145 mg, 1.44 mmol) in THF (1 mL) was added and the reaction mixture was allowed to warm to room temperature and stirred for 12 h. The reaction mixture was concentrated to dryness and the crude residue was purified by preparative TLC to give Compound 339. MS (ESI) calcd for C₂₁H₁₇F₃N₄O₃: 430.1. found: 431 [M+H].

Example 6 Synthesis of 3-methyl-4-oxo-N-(pyridin-2-yl)-2-(2-((trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 212) Step 1. Preparation of methyl 2-amino-3-(pyridin-2-ylcarbamoyl)benzoate (22)

A solution of 2-amino-3-(methoxycarbonyl)benzoic acid 20 (1 g, 5.1 mmol), 2-aminopyridine 21 (723 mg, 7.7 mmol), HOBT (1.39 g, 10.3 mmol), EDCI (1.9 g, 10.3 mmol) and DMAP (1.25 g, 10.3 mmol) in DMF (40 mL) was stirred at 70° for 12 h. The reaction mixture was poured into H2O. The resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 22 (0.9 g, 58% yield).

Step 2. Preparation of 2-amino-3-(pyridin-2-ylcarbamoyl)benzoic acid (22)

LiOH.H₂O (1.8 g, 42.8 mmol) was added to a solution of methyl 2-amino-3-(pyridin-2-ylcarbamoyl)benzoate 22 (2.9 g, 10.7 mmol) in THF (30 mL) and H₂O (30 mL). The reaction mixture was heated at 60° for 5 h. The THF was removed en vacuo and H₂O (10 mL) was added. The pH was adjusted to 4 using aq HCl and the resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 23 (2.1 g, 76% yield).

Step 3. Preparation of 4-oxo-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazine-8-carboxamide (25)

2-(trifluoromethyl)benzoyl chloride 24 (180 mg, 0.88 mmol) was added to a suspension of 2-amino-3-(pyridin-2-ylcarbamoyl)benzoic acid 23 (150 mg, 0.58 mmol) in pyridine (8 mL). The reaction mixture was stirred at room temperature for 12 h then poured into ice cooled H₂O. The resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 25 (90 mg, 38% yield) as a white solid.

Step 4. Preparation of 3-methyl-4-oxo-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 212)

A solution of 30 wt % methylamine (7 mL) in MeOH and 4-oxo-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazine-8-carboxamide 25 (90 mg, 0.22 mmol) was heated under gentle reflux for 1 h then cooled to room temperature. The resulting precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give Compound 212 (20 mg, 22% yield) as a white solid. MS (ESI) calc. for C₂₂H₁₅F₃N₄O₂: 424.11. found: 425 [M+H].

Example 7 Synthesis of 3-methyl-4-oxo-N-(pyridin-2-yl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 275) Step 1. Preparation of 2-(4-bromophenyl)-4-oxo-N-(pyridin-2-yl)-4H-benzo[d][1,3]oxazine-8-carboxamide (27)

2-(4-bromophenyl)-4-oxo-N-(pyridin-2-yl)-4H-benzo[d][1,3]oxazine-8-carboxamide 27 was prepared by a procedure similar to that reported for 4-oxo-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazine-8-carboxamide 25 in 91% yield

Step 2. Preparation of 2-(4-bromophenyl)-3-methyl-4-oxo-N-(pyridin-2-yl)-3,4-dihydroquinazoline-8-carboxamide (28)

Compound 28 was prepared by a procedure similar to that reported for 3-methyl-4-oxo-N-(pyridin-2-yl)-2-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide Compound 212 in 76% yield

Step 3. Preparation of 3-methyl-4-oxo-N-(pyridin-2-yl)-2-(4-(pyrrolidin-1-ylmethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 275)

Degassed THF/H₂O (4 mL, 10:1) was added to a microwave vial containing 2-(4-bromophenyl)-3-methyl-4-oxo-N-(pyridin-2-yl)-3,4-dihydroquinazoline-8-carboxamide 28 (100 mg, 0.23 mmol), Cs₂CO₃ (224 mg, 0.69 mmol), Pd(OAc)₂ (1.5 mg, 0.007 mmol), XPHOS (65 mg, 0.014 mmol), and potassium 1-trifluoroboratomethylpyrrolidine (48 mg, 0.25 mmol). The reaction mixture was heated at 150° C. in a microwave reactor for 30 min., poured into H₂O and extracted with EtOAc. The combined organics were washed with brine, dried, concentrated and the crude residue purified by MPLC eluting with CH₂Cl₂/MeOH (0-10%) to give Compound 275 (63 mg, 63% yield). MS (ESI) calc. for C₂₆H₂₅N₅O₂: 439.20. found: 440 [M+H].

Example 8 Synthesis of N-(3,4-dimethoxyphenyl)-3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 220)

A microwave vial was charged with isatoic anhydride-2-carboxylic acid 29 (Clark, et al. J. Med. Chem. 1995, 38, 1493-1504) (115 mg, 0.555 mmol), 3,4-dimethoxyaniline 30 (102 mg, 0.666 mmol) and pyridine (2.0 mL). The reaction mixture was heated at 200° C. in a microwave for 2 h. Upon cooling, 3-(trifluoromethyl)benzoyl chloride 2 (120 ml, 0.800 mmol) was added, and the vial heated to 100° C. for 1 h. After cooling, a solution of methylamine (1.0 m, 2.0 mmol) in MeOH was added, and the reaction heated to 100° C. for an additional 1 h. The volatiles were removed in vacuo and the crude residue was purified by MPLC to give Compound 220 (69 mg, 26% yield). MS (ESI) calc. for C₂₅H₂₀F₃N₃O₄: 483.14. found: 484 [M+H].

Example 9 Synthesis of 3-methyl-4-oxo-N-(6-(pyrrolidin-1-yl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound 350) Step 1. Preparation of 2-amino-3-(methylcarbamoyl)benzoic acid (31)

Aq NaOH (23.4 mL, 46.8 mmol) was added to a solution of methylamine hydrochloride (3.52 g, 53.7 mmol) in H₂O (37 mL). 2,4-dioxo-2,4-dihydro-1H-benzo[d][1,3]oxazine-8-carboxylic acid 29 (3.23 g, 15.6 mmol) was in portions. After addition was complete, the solution was stirred at ambient temperature for 1.5 h. 6M HCl was added until pH=3. The resulting precipitate was collected by filtration, rinsed with H₂O and dried to give 31 (2.60 g, 86% yield).

Step 2. Preparation of 3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxylic acid (33)

Sodium metabisulfite (3.56 g, 18.7 mmol) was added to a solution of 2-amino-3-(methylcarbamoyl)benzoic acid 31 (2.80 g, 14.4 mmol) and 3-(trifluoromethyl)benzaldehyde 32 (2.51 g, 14.4 mmol) in DMAC (45 mL). The reaction mixture was stirred at 100° C. for 21 h. H₂O (150 mL) was added and the resulting precipitate was collected by filtration, rinsed with H₂O and dried to give 33 (4.31 g, 84% yield).

Step 3. Preparation of 3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carbonyl chloride (34)

A solution of thionyl chloride (10 mL) and 3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxylic acid 33 (1.00 g, 2.87 mmol) was heated at reflux for 1 h. Upon cooling to room temperature, all volatiles were removed under vacuum, to give 34 (1.05 g, 100% yield) as a white solid.

Step 4. Preparation of 3-methyl-4-oxo-N-(6-(pyrrolidin-1-yl)pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carboxamide (Compound)

Triethylamine (100 μL) and 6-pyrollidin-1-ylpyridin-2-amine 35 (0.136 mmol) was added to a solution of 3-methyl-4-oxo-2-(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-8-carbonyl chloride 34 (50 mg, 0.136 mmol) in dioxane (1 mL). The reaction was stirred at 70° C. for 2 d, after which 1N HCl (4 mL) was added. The resulting precipitate was collected by filtration, rinsed with 5 mL H₂O, 5 mL pentane, and dried under vacuum to give Compound 350 (40 mg, 56% yield). MS (ESI) calc. for C₂₆H₂₂F₃N₅O₂: 493.17. found: 494 [M+H].

Example 10 Synthesis of N-(4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinolin-8-yl)pyrazine-2-carboxamide (Compound 409) Step 1. Preparation of ethyl 8-nitro-4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-3-carboxylate (36)

A solution of diisopropylamine (7.00 mL, 50.0 mmol) in THF (100 mL) was cooled to −78° C., and n-BuLi (20.0 mL, 2.5 M in hexanes, 50.0 mmol) was added. After stirring for 20 min at −78° C., ethyl acetate (5.72 mL, 58.6 mmol) was added. The solution was stirred at −78° C. for 10 min, warmed to 0° C. and stirred for 10 min, then cooled back to −78° C. A solution of 8-nitro-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazin-4-one 3 (9.85 g, 29.3 mmol) in THF (100 mL) was added over 5 min to the LDA/EtOAc mixture. The reaction was stirred at −78° C. for 1 h, then warmed to room temperature over 2 h. 1N NaOH (50 mL) was then added, and the reaction stirred vigorously overnight. Brine (50 mL) was added, and the organic layer separated. The aqueous layer was extracted with ethyl acetate (2×100 mL), and the combined organic layers dried with MgSO₄ and concentrated under reduced pressure. The remaining material was purified by MPLC (using a gradient of 20% to 50% EtOAc in pentane) to give 36 (5.00 g, 42% yield).

Step 2. Preparation of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinolin-4(1H)-one (37)

A microwave vial was charged with of 8-nitro-4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-3-carboxylate 36 (1.0 g, 2.46 mmol), dioxane (8 mL) and 1N HCl (4 mL). The reaction mixture was heated at 190° C. in the microwave for 50 min. The volatiles were evaporated and the residue dried under vacuum to give 37 (707 mg, 86% yield).

Step 3. Preparation of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate (38)

A solution of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinolin-4(1H)-one 37 (707 mg, 2.11 mmol), triethylamine (1 mL), and Ac₂O (0.7 mL) in CH₂Cl₂ (10 mL) was stirred at room temperature for 16 h. Sat. aq NaHCO₃ (10 mL) was added, and the reaction extracted with CH₂Cl₂. The combined organic layers were dried (MgSO₄) and concentrated to give 38.

Step 4. Preparation of 8-amino-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate (39)

Pd/C (300 mg) was added to a solution of 8-nitro-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate 38 (2.11 mmol) in ethyl acetate (10 mL). Hydrogen gas was bubbled through the reaction for 10 min, and then the reaction was stirred under 1 atm. hydrogen for 16 h. The catalyst was removed by filtration through 10 g of silica. The solvents were removed in vacuo to give 39 (400 mg, 55% yield for both steps).

Step 5. Preparation of 8-(pyrazine-2-carboxamido)-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate (41)

Pyrazine-2-carbonyl chloride 40 (100 mg) was added to a solution of 8-amino-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate 39 (50 mg, 0.145 mmol) and triethylamine (0.20 mL) in dichloromethane (3 mL). The reaction mixture was stirred at 40° C. for 16 h. The reaction mixture was poured into dilute NaHCO₃ (10 mL), and extracted with 3×30 mL dichloromethane. The combined organic layers were dried (MgSO₄) and the solvent removed in vacuo. The crude residue was purified by MPLC (97:3 dichloromethane:methanol) to give 41 (33 mg, 50% yield).

Step 6. Preparation of N-(4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinolin-8-yl)pyrazine-2-carboxamide (Compound 409)

A solution of 8-(pyrazine-2-carboxamido)-2-(3-(trifluoromethyl)phenyl)quinolin-4-yl acetate 41 (33 mg, 0.073 mmol) in THF (2 mL) and 1N NaOH (0.5 mL) was heated at 60° C. for 48 h. The reaction mixture was cooled to room temperature and 1N HCl (5 mL) was added. The resulting precipitate was collected by filtration, rinsed with 3 mL H₂O, and dried under vacuum to give Compound 409 (21 mg, 70% yield). MS (ESI) calc. for C₂₁H₁₃F₃N₄O₂: 410.10. found: 411 [M+H].

Example 11 Synthesis of 4-oxo-N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-8-carboxamide (Compound 401) Step 1. Preparation of methyl 4-oxo-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazine-8-carboxylate (42)

To a solution of 2-amino-3-(methoxycarbonyl)benzoic acid 20 (1.16 g, 5.94 mmol) in pyridine (10 mL) was added 3-trifluoromethylbenzoyl chloride 2 (0.90 mL, 5.94 mmol). The reaction was stirred for 16 h, poured into sat. aq NaHCO₃ (50 mL), extracted with dichloromethane (3×50 mL) and the combined organic layers were dried (MgSO₄) and concentrated. Toluene (10 mL) was added and the solution was concentrated in vacuo. The crude residue was purified by MPLC eluting with pentane/EtOAc (10-50%) to give 42 (1.45 g, 70% yield).

Step 2. Preparation of 3-ethyl 8-methyl 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-3,8-dicarboxylate (43)

n-BuLi (4.4 mL, 2.5 M in hexanes, 11.0 mmol) was added to a solution of diisopropylamine (1.57 mL, 11.2 mmol) in THF (30 mL) at −78° C. After stirring for 20 min, EtOAc (1.09 mL, 11.2 mmol) was added. The solution was stirred at −78° C. for 10 min, warmed to 0° C., stirred for 10 min, then cooled back to −78° C. A solution of 4-oxo-2-(3-(trifluoromethyl)phenyl)-4H-benzo[d][1,3]oxazine-8-carboxylate 42 (2.00 g, 5.72 mmol) in THF (50 mL) was added over 5 min to the reaction mixture and stirring at −78° C. continued for 1 h. The reaction mixture was then allowed to warm to room temperature over 2 h. MeOH (50 mL) was added followed by solid NaOMe (1.62 g, 30 mmol), and the reaction stirred vigorously for 12 h at room temperature. The solution was concentrated to 20 mL total volume in vacuo, H₂O (100 mL) was added, and the mixture was extracted with ethyl acetate (2×100 mL). The combined organic layers were dried (MgSO₄) and concentrated in vacuo. The crude residue was purified by MPLC eluting with pentane/EtOAc (10-50%) to give 43 (950 mg, 39% yield).

Step 3. Preparation of 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-8-carboxylic acid (44)

A 20 mL microwave vial was charged with 3-ethyl 8-methyl 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-3,8-dicarboxylate 43 (820 mg, 1.96 mmol), dioxane (8 mL) and 1N HCl (4 mL). The reaction mixture was heated in a microwave reactor at 200° C. for 25 min then concentrated to dryness in vacuo to give 44 (585 mg, 90% yield).

Step 4. Preparation of 4-oxo-N-(pyridin-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-8-carboxamide (Compound 401)

A mixture of 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydroquinoline-8-carboxylic acid 44 (250 mg, 0.750 mmol), HATU (570 mg, 1.5 mmol), 2-aminopyridine 21 (141 mg, 1.5 mmol) and diisopropylethylamine (392 μl, 2.25 mmol) in DMAC (5 mL) was heated at 75° C. for 16 h. The reaction mixture was poured in H₂O (25 mL) and the resulting precipitate was collected by filtration. The crude residue was purified by MPLC Compound 401 (102 mg, 33% yield). MS (ESI) calc. for C₂₂H₁₄F₃N₃O₂: 409.10. found: 410 [M+H].

Example 12 Synthesis of 4-oxo-N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-8-carboxamide (Compound 449) Step 1. Preparation of 4-amino-5-bromonicotinic acid (46)

A sealed tube was charged with 4-aminonicotinic acid 45 (8.00 g, 57.9 mmol), acetic acid (75 mL) and water (75 mL). The reaction was heated at 75° C. and stirred vigorously until homogeneous. After cooling to 50° C., bromine (10.0 mL, 195 mmol) was added and stirring continued for 16 h. Upon cooling to 0° C., the resulting orange precipitate was collected by filtration, rinsed with H₂O, and dried under vacuum to give 46 (10.5 g, 84% yield).

Step 2. Preparation of 8-bromo-2-(3-(trifluoromethyl)phenyl)-4H-pyrido[4,3-d][1,3]oxazin-4-one (47)

A mixture of 5-bromo-4-aminonicotinic acid 46 (10.5 g, 48.4 mmol) and pyridine (60 mL) was heated at 60° C. until homogeneous. 3-(trifluoromethyl)benzoyl chloride 2 (8.0 mL, 53.0 mmol) was added and stirring continued for 2 h. H₂O (200 mL) was added and the mixture cooled to 0° C. The resulting yellow precipitate was collected by filtration, rinsed with H₂O (100 mL) then pentane (100 mL), and dried under vacuum to give the 47 (8.50 g, 47% yield).

Step 3. Preparation of ethyl 8-bromo-4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-3-carboxylate (48)

A mixture of THF (15 mL) and diisopropylamine (1.40 mL, 10.0 mmol) was cooled to −78° C., and n-BuLi (4.0 mL, 2.5 M in hexanes, 10.0 mmol) was added. After stirring at −78° C. for 20 min, ethyl acetate (1.0 L, 10.2 mmol) was added and stirring was continued for 5 min. 8-bromo-2-(3-(trifluoromethyl)phenyl)-4H-pyrido[4,3-d][1,3]oxazin-4-one 47 (990 mg, 2.67 mmol) was added. After 1 h, the reaction mixture was gradually warmed to room temperature. 1N NaOH (15 mL) was added and the reaction mixture was stirred for 24 h. The mixture was poured into brine (50 mL), extracted with ethyl acetate (2×50 mL), dried (MgSO₄), and concentrated in vacuo. The resulting solid was purified by MPLC to give 48 (910 mg, 77% yield).

Step 4. Preparation of 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-8-carboxylic acid (49)

A mixture of ethyl 8-bromo-4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-3-carboxylate 48 (910 mg, 2.06 mmol), palladium acetate (22.4 mg, 0.100 mmol), XPhos (48.8 mg, 0.102 mmol), copper (I) cyanide (450 mg, 5.02 mmol), and sodium carbonate (636 mg, 6.00 mmol) in DMF (7.0 mL) was heated at 120° C. for 16 h. After cooling to room temperature, the mixture was poured into methanol (100 mL), filtered through celite, and concentrated in vacuo. The crude residue was dissolved in dioxane (10 mL) and 1N HCl (5 mL). The reaction mixture was heated at 190° C. for 50 min in a microwave reactor. The volatiles were removed in vacuo and the residue was redissolved in methanol (10 mL) and 1N NaOH (5 mL). This solution was heated in a microwave reactor at 160° C. for 1.5 h, after which 6N HCl (5 mL) was added. The resulting precipitate was collected by filtration, rinsed with H₂O and dried under vacuum to give 49 (400 mg, 58% yield.)

Step 5. Preparation of 4-oxo-N-(thiazol-2-yl)-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-8-carboxamide (Compound 449)

A mixture of 4-oxo-2-(3-(trifluoromethyl)phenyl)-1,4-dihydro-1,6-naphthyridine-8-carboxylic acid 49 (100 mg, 0.299 mmol) and carbonyldiimidazole (100 mg, 0.617 mmol) in dioxane (2 mL) was heated at 70° C. for 1 h. 2-aminothiazole 50 (120 mg, 1.20 mmol) was added and the reaction stirred at 70° C. for 16 h. Upon cooling to room temperature, 1N HCl (5 mL) was added and the resulting precipitate was collected by filtration, rinsed with H₂O (5 mL), and purified by preparative HPLC to give Compound 449 as the trifluoroacetate salt (4.0 mg, 2.5% yield).

Additional compounds of the invention were synthesized in a similar fashion by reacting the appropriate carboxylic acid with appropriate amine. The synthesis of various amine and carboxylic acid intermediates present in the compounds of this invention are shown in Examples 13-51, below.

Example 13 Synthesis of 2-(difluoromethyl)benzoyl chloride 54 Step 1. Preparation of methyl 2-(difluoromethyl)benzoate (52)

A solution of methyl 2-formylbenzoate 51 (10 g, 61 mmol) and bis-(2-methoxyethyl)amino-sulfur trifluoride (40.4 g, 183 mmol) in CH₂Cl₂ was heated at reflux for 12 hours. The reaction mixture was cooled to room temperature, concentrated and partitioned between EtOAc (500 mL)/H₂O (300 mL). NaHCO₃ was added to adjust the pH to 8. The organic phase was separated, washed with brine, dried and concentrated. The residue was purified by flash chromatography to give 52 (7 g, 62% yield)

Step 2. Preparation of 2-(difluoromethyl)benzoic acid (53)

A mixture of methyl 2-(difluoromethyl)benzoate 52 (7 g, 38 mmol) and 10% aq. NaOH (100 mL) in MeOH (50 mL) was heated at reflux for 30 min. The pH was adjusted to 4 by the addition of 3N HCl. The resulting solid was collected by filtration, rinsed with H₂O and dried to give 53 (6 g, 93% yield).

Step 3. Preparation of 2-(difluoromethyl)benzoyl chloride (54)

A solution of 2-(difluoromethyl)benzoic acid 53 (1.8 g, 10 mmol) in thionyl chloride (25 ml) was heated at reflux for 3 h. The reaction mixture was concentrated and dried under vacuum to give 54. The crude acid chloride was used without further purification.

Example 14 Synthesis of 3-(difluoromethyl)benzoyl chloride (56)

Compound 56 was prepared by a procedure similar to that reported for 2-(difluoromethyl)benzoyl chloride 54 in 32% yield.

Example 15 Synthesis of 6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)picolinic acid (59)

Solketal 58 (23.5 g, 178 mmol) was added to a suspension of NaH (7.1 g, 178 mmol, 60 wt % dispersion in mineral oil) in THF (400 mL) cooled to 0° C. The reaction mixture was stirred at room temperature for 1 h and 6-bromopicolinic acid 57 (12 g, 59.4 mmol) was added. The reaction mixture was heated at reflux for 1.5 h. After cooling to room temperature, H₂O (50 mL) was added and the pH was adjusted to 3 by the addition of 3 N HCl. The mixture was poured into brine and extracted with EtOAc. The combined organics were dried (Na₂SO₄) and concentrated. The crude product was recrystallized from pentane/EtOAc to give 59 (10 g, 66% yield). The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 16 Synthesis of 2((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)nicotinic acid (61)

Compound 61 was prepared by a procedure similar to that reported for 6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)picolinic acid 59 in 23% yield. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 17 Synthesis 6-(morpholinomethyl)picolinic acid 65 Step 1. Preparation of 4-((6-bromopyridin-2-yl)methyl)morpholine (64)

NaBH(OAc)₃ (68.5 g, 0.323 mol) was added to a solution of 6-bromopicolinaldehyde 62 (40 g, 0.22 mol) and morpholine 63 (20.9 g, 0.24 mol) in 1,2-dichloroethane (500 mL). The mixture was stirred at room temperature for 16 h. Saturated NaHCO₃ (500 mL) was added and the mixture was extracted with EtOAc, washed with brine, dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with petroleum ether:ethyl acetate (10:1) to give 64 (38 g, 68% yield).

Step 2. Preparation of 6-(morpholinomethyl)picolinic acid (65)

n-BuLi (56 mL, 0.140 mol) in THF was added to a solution of 4-((6-bromopyridin-2-yl)methyl)morpholine 64 (30 g, 0.12 mol) in THF (500 mL) at −78° C. The mixture was stirred for 30 min and CO₂ (gas) was bubbled through the reaction mixture for 30 min. The volatiles were removed in vacuo and the residue was extracted with CH₂Cl₂/MeOH (1:1). The solvent was evaporated and the residue was washed with CH₂Cl₂ to give 65 (11.0 g, 42% yield).

Example 18 Synthesis of 6-(pyrrolidin-1-ylmethyl)picolinic acid 70 Step 1. Preparation of methyl 6-(chloromethyl)picolinate (67)

SOCl₂ (57 g, 0.48 mol) was added to a solution of methyl 6-(hydroxymethyl)picolinate 66 (40.0 g, 0.239 mol) (Chem. Eur. J. 2006, 12, 6393-6402) in dichloromethane (500 mL) at room temperature. The mixture was stirred at 40° C. for 1 h and sat. aq K₂CO₃ was added to adjust the pH to 9. The mixture was extracted with CH₂Cl₂ and the combined organics were washed with brine, dried (Na₂SO₄), and concentrated in vacuo to give 67 (45 g).

Step 2. Preparation of methyl 6-(pyrrolidin-1-ylmethyl)picolinate (69)

K₂CO₃ (66 g, 0.48 mol) was added to a solution of methyl 6-(chloromethyl)picolinate 67 (45.0 g) and pyrrolidine 68 (34 g, 0.48 mol) in DMF (300 mL). The reaction mixture was heated at 80° C. for 12 h. H₂O (300 mL) was added and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried (Na₂SO₄) and concentrated in vacuo to give 69 (36 g).

Step 3. Preparation of 6-(pyrrolidin-1-ylmethyl)picolinic acid (70)

A mixture of methyl 6-(pyrrolidin-1-ylmethyl)picolinate 69 (36 g) and NaOH (40 g, 1.0 mol) in ethanol/H₂O (320 mL) was stirred at 75° C. for 16 h. The pH was adjusted to 7 with 3N HCl and extracted with EtOAc. The aqueous layer was concentrated to dryness and extracted with dichloromethane/methanol (v:v=3:1), The organic layer was dried to give 70 (27 g, 55% yield).

Example 19 Synthesis of N-methyl proline 72

Compound 72 was prepared by a procedure similar to that reported in J. Org. Chem. 2003, 66, 2652.

Example 20 Synthesis of 1-methyl-5-oxopyrrolidine-2-carboxylic acid 73

Compound 73 was prepared by a procedure similar to that reported in J. Heterocyclic. Chem. 1991, 28, 1143.

Example 21 Synthesis of 3-(morpholinomethyl)aniline 74

Compound 74 was prepared by a procedure similar to that reported in J. Med. Chem. 1990, 33(1), 327-36.

Example 22 Synthesis of 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 81 Step 1. Preparation of ethyl 6-aminopicolinate (76)

To a solution of 2-amino-6-pyridinecarboxylic acid 75 (6.0 g, 43.5 mmol) in ethanol (150 mL) was added thionyl chloride (12.0 g, 101 mmol) at 0° C. The resulting reaction mixture was stirred at reflux for 12 h. Upon cooling to room temperature, the reaction mixture was concentrated under reduced pressure. Saturated aqueous Na₂CO₃ solution was added until the pH of the solution reached 9. The mixture was concentrated under reduced pressure and dichloromethane (150 mL) was added to the resulting residue. The mixture was stirred vigorously at room temperature for 30 min and then filtered. The filtrate was concentrated under reduced pressure to afford 76 (5.5 g, 76% yield).

Step 2. Preparation of ethyl 6-(tert-butoxycarbonylamino)picolinate (77)

To a solution of ethyl 6-aminopicolinate 76 (5.5 g, 33 mmol) in t-BuOH (120 mL) and acetone (40 mL) was added 4-dimethylaminopyridine (0.08 g, 0.66 mmol) and di-tert-butyl dicarbonate (10.8 g, 49.5 mmol). The reaction mixture was stirred at room temperature for 18 h. The solvent was removed by concentration under reduced pressure and a mixture of hexane/dichloromethane (180 mL, 3:1) was added. The resulting mixture was cooled to −20° C. for 2 h. The resulting solids were collected by filtration and dried to afford 77 (11.0 g, 91% yield).

Step 3. Preparation of tert-butyl 6-(hydroxymethyl)pyridin-2-ylcarbamate (78)

To a stirred solution of ethyl 6-(bis(tert-butoxycarbonyl)amino)picolinate 77 (11.0 g, 33 mmol) in THF (120 mL) under nitrogen was added LiAlH₄ (3.80 g, 100 mmol) in THF (60 mL) over a period of 30 min at 0° C. The reaction mixture was stirred at 0° C. for 6 h and carefully quenched by the addition of H₂O (2.0 mL) and 10% NaOH solution (4.0 mL) at 0° C. The reaction mixture was filtered and the filtrate was dried (Na₂SO₄) and concentrated under reduced pressure. The resulting residue purified by chromatography (1:1 petroleum ether:ethyl acetate) to afford 78 (3.0 g, 41% yield).

Step 4. Preparation of (6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate (79)

To a solution of tert-butyl 6-(hydroxymethyl)pyridin-2-ylcarbamate 78 (3.0 g, 13.4 mmol) and diisopropylethylamine (5.0 g, 40 mmol) in acetonitrile (30 mL) was added methanesulfonyl chloride (2.0 g, 17.4 mmol) over a period of 30 min at 0° C. and the mixture was stirred for 2 h at room temperature. The reaction was quenched by adding saturated aqueous NaHCO₃ and extracted with ethyl acetate (3×60 mL). The combined organic layers were washed with brine, dried (Na₂SO₄) and concentrated under reduced pressure to afford quantitative yield of crude 79.

Step 5. Preparation of tert-butyl 6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate (80)

A mixture of (6-(tert-butoxycarbonylamino)pyridin-2-yl)methyl methanesulfonate 79 (1.30 g, 3.2 mmol), pyrrolidine 68 (0.46 g, 6.4 mmol) and K₂CO₃ (1.30 g, 9.6 mmol) in acetonitrile (15 mL) was stirred at room temperature for 12 h. Saturated aqueous NaHCO₃ was added and the mixture was concentrated under reduced pressure. The resulting aqueous layer was extracted with EtOAc. The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure to afford 80 (0.75 g, 62% yield).

Step 6. Preparation of 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine (81)

To a solution of tert-butyl 6-(pyrrolidin-1-ylmethyl)pyridin-2-ylcarbamate 80 (750 mg, 2.71 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (4.0 mL) at room temperature. The resulting reaction mixture was stirred at room temperature for 6 h and then concentrated under reduced pressure. Saturated aqueous Na₂CO₃ was added to the resulting residue until the solution pH reached 9. The mixture was then extracted with ethyl acetate (3×25 mL). The combined organic layers were dried with Na₂SO₄ and concentrated under reduced pressure to afford 81 (440 mg, 92% yield).

Example 23 Synthesis of 6-(morpholinomethyl)pyridin-2-amine 82

Compound 82 was prepared by a method similar to that reported for 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 81.

Example 24 Synthesis of (R)-6-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine 83

Compound 83 was prepared by a method similar to that reported for 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 81.

Example 25 Synthesis of (S)-6-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine 84

Compound 84 was prepared by a method similar to that reported for 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 81.

Example 26 Synthesis of 6-(piperazin-1-ylmethyl)pyridin-2-amine 85

Compound 85 was prepared by a method similar to that reported for 6-(pyrrolidin-1-ylmethyl)pyridin-2-amine 81.

Example 27 Synthesis of tert-butyl 4-((6-aminopyridin-2-yl)methyl)piperazine-1-carboxylate 86

To a solution of 6-(piperazin-1-ylmethyl)pyridin-2-amine 85 in THF was added di-tert-butyl carbonate (1 eq) and 4-(dimethyl)aminopyridine (catalytic). The reaction mixture was stirred at room temperature for 18 h. It was then concentrated under reduced pressure. Pentane was added and the resulting solid was collected by filtration and dried to afford 86. The Boc protecting group could be removed after coupling with the appropriate carboxylic acid by treatment with TFA/CH₂Cl₂ for 12 h.

Example 28 Synthesis of 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate 91 Step 1. Preparation of ethyl 2-(tert-butoxycarbonylamino)thiazole-4-carboxylate (88)

Ethyl 2-aminothiazole-4-carboxylate 87 (10.0 g, 58.1 mmol) was taken up in 150 mL of anhydrous THF along with di-tert-butyl carbonate (12.67 g, 58.1 mmol) and 4-(dimethyl)aminopyridine (DMAP) (10 mg, 0.082 mmol). The reaction mixture was stirred at 50° C. for 4 h and then at room temperature for 18 h. It was then concentrated under reduced pressure to obtain a thick oil. Pentane was added and the resulting crystalline materials were collected by filtration and dried to afford 88 (10.5 g, 66% yield).

Step 2. Preparation of tert-butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate (89)

Ethyl 2-(tert-butoxycarbonylamino)thiazole-4-carboxylate 88 (10.5 g, 38.6 mmol) was dissolved in 300 mL of anhydrous THF and cooled in dry ice-acetonitrile bath. A solution of 1 M Super Hydride™ in THF (85 mL) was then added over a period of 10 min. The resulting reaction mixture was stirred at −45° C. for 2 h. Another portion of 1 M Super Hydride™ in THF (35 mL) was then added and the reaction mixture was stirred for an additional 2 h at −45° C. The reaction was quenched at −45° C. by the addition of 50 mL of brine. Upon warming to room temperature, the reaction mixture was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried with Na₂SO₄, and concentrated under reduced pressure. The resulting residue was purified by chromatography to afford 89 (6.39 g, 72% yield).

Step 3. Preparation of tert-butyl 4-(morpholinomethyl)thiazol-2-ylcarbamate (90)

tert-Butyl 4-(hydroxymethyl)thiazol-2-ylcarbamate 89 (2.00 g, 8.68 mmol) was taken up in 25 mL of CH₂Cl₂ along with Et₃N (1.82 mL, 13.05 mmol) and cooled to 0° C. Methanesulfonyl chloride (0.85 mL, 10.88 mmol) was added and the resulting reaction mixture was stirred at 0° C. for 60 min. Morpholine 63 (3.0 mL, 35 mmol) was then added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated under reduced pressure. The resulting residue was taken up in EtOAc and washed with dilute aqueous NaHCO₃, brine, dried with Na₂SO₄, and concentrated under reduced pressure. This material was purified by filtering through a short column of silica gel. The filtrate was concentrated to afford 90 (1.88 g, 69% yield).

Step 4. Preparation of 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate (91)

Tert-butyl 4-(morpholinomethyl)thiazol-2-ylcarbamate 90 (1.88 g, 6.28 mmol) was treated with 20 mL of 25% trifluoroacetic acid in CH₂Cl₂ for 18 h at room temperature. After all the solvent had been removed by concentrating and drying under high vacuum, the resulting residue was treated with a mixture of pentane/EtOAc to afford 91 (1.96 g, 100% yield) as a white solid.

Example 29 Synthesis of 4-(pyrrolidin-1-ylmethyl)thiazol-2-amine trifluoroacetate 92

Compound 92 was prepared by a procedure similar to that reported for 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate 91.

Example 30 Synthesis of 5-(morpholinomethyl)thiazol-2-amine trifluoroacetate 93

Compound 93 was prepared by a procedure similar to that reported for 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate 91.

Example 31 Synthesis of 5-(pyrrolidin-1-ylmethyl)thiazol-2-amine trifluoroacetate 94

Compound 94 was prepared by a procedure similar to that reported for 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate 91.

Example 32 Synthesis of 4-(piperazin-1-ylmethyl)thiazol-2-amine trifluoroacetate 95

Compound 95 was prepared by a procedure similar to that reported for 4-(morpholinomethyl)thiazol-2-amine trifluoroacetate 91.

Example 33 Synthesis of tert-butyl 4-((2-aminothiazol-4-yl)methyl)piperazine-1-carboxylate 96

Compound 96 was prepared by a procedure similar to that reported for tert-butyl 4-((6-aminopyridin-2-yl)methyl)piperazine-1-carboxylate 86. The Boc protecting group could be removed after coupling with the appropriate carboxylic acid by treatment with TFA/CH₂Cl₂ for 12 h.

Example 34 Synthesis of 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrimidin-4-amine 98

To a solution of solketal 58 (34.4 g, 260 mmol) in THF (150 mL) was added NaH (10.4 g, 260 mmol) at room temperature and the mixture stirred for 1 h. 2-chloro-4-aminopyrimidine 97 (15.0 g, 115 mmol) was then added, and the mixture was stirred at 70° C. for 48 h. The reaction mixture was concentrated and the crude residue was purified by flash chromatography (dichloromethane:methanol=15:1-10:1) to give 98 (18.2 g, 70% yield) as an oil. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 35 Synthesis of 6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrazin-2-amine 99

Compound 99 was prepared by a method similar to that reported for 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrimidin-4-amine 98. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 36 Synthesis of (S)-6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-aminopyridine 100

Compound 100 was prepared by a method similar to that reported for 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrimidin-4-amine 98. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 37 Synthesis of (R)-6-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-2-aminopyridine 101

Compound 101 was prepared by a method similar to that reported for 2-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)pyrimidin-4-amine 98. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 38 Synthesis of (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 105 Step 1. Preparation of (R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane (104)

A mixture of 3-nitrophenol 102 (2.00 g, 14.4 mmol), potassium carbonate (4.96 g, 35.9 mmol) and (S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane 103 (2.55 mL, 18.7 mmol) in DMF (20 mL) was heated in a microwave reactor at 160° C. for 4 h. The crude reaction mixture was poured into H₂O and extracted with dichloromethane (3×15 mL). The combined organic layers were dried (Na₂SO₄) and concentrated under reduced pressure. The crude residue was purified by chromatography using ethyl acetate: pentane to obtain (R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane 104 (1.90 g, 52% yield) as an amber-colored oil.

Step 2. Preparation of (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline (105)

A mixture of Fe powder (2.38 g, 42.5 mmol), NH₄Cl (2.27 g, 42.5 mmol) and (R)-2,2-dimethyl-4-((3-nitrophenoxy)methyl)-1,3-dioxolane 104 (1.80 g, 7.09 mmol) in isopropanol (30 mL)/H₂O (10 mL) was heated at reflux for 18 h. The crude material was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The resulting aqueous layer was extracted with dichloromethane (3×15 mL). The combined organic layers were dried with Na₂SO₄, and concentrated under reduced pressure to afford 105 (1.25 g, 76% yield). The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 39 Synthesis of 3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 106

Compound 106 was prepared by a method similar to that reported for (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 105. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 40 Synthesis of (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 107

Compound 107 was prepared by a method similar to that reported for (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 105. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 41 Synthesis of 4((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 108

Compound 108 was prepared by a method similar to that reported for (R)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)aniline 105. The acetonide protecting group could be removed after coupling with the appropriate aniline by treatment with EtOH/3 N HCl (4:1) at reflux for 12 h.

Example 42 Synthesis of 2-(pyrrolidin-1-yl)pyridin-4-amine 110

A mixture of 2-chloro-4-aminopyridine 109 (2.29 g, 17.8 mmol) and pyrrolidine 68 (5.0 mL) was heated at 200° C. in a microwave reactor for 10 min. After cooling to room temperature, the solid was filtered and washed with dichloromethane (10 mL×3). The filter cake was dissolved in aqueous K₂CO₃ and extracted with CH₂Cl₂ (40 mL×3). The combined organic layers were dried over Na₂SO₄ and concentrated to obtain 110 (2.30 g, 79% yield).

Example 43 Synthesis of 2-morpholinopyridin-4-amine 111

Compound III was prepared by a method similar to that reported for 2-(pyrrolidin-1-yl)pyridin-4-amine 110.

Example 44 Synthesis of 6-morpholinopyridin-2-amine 112

Compound 112 was prepared by a method similar to that reported for 2-(pyrrolidin-1-yl)pyridin-4-amine 110.

Example 45 Synthesis of 6-(pyrrolidin-1-yl)pyridin-2-amine 35

Compound 35 was prepared by a method similar to that reported for 2-(pyrrolidin-1-yl)pyridin-4-amine 110.

Example 46 Synthesis of (S)-5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine 120 Step 1. Preparation of ethyl 6-aminonicotinate (114)

To a solution of 2-amino-5-pyridinecarboxylic acid 113 (150 g, 1.09 mol) in ethanol (2 L) was added thionyl chloride (259 g, 2.18 mol) at 0° C. The mixture was heated at reflux for 12 h. The solvent was removed under reduced pressure. Saturated aq Na₂CO₃ was added to adjust the pH to 9 and the resulting solid was collected by filtration, rinsed with H₂O, and dried to give 114 (160 g, 88% yield).

Step 2. Preparation of ethyl 6-(bis(tert-butoxycarbonyl)amino)nicotinate (115)

To a solution of ethyl 6-aminonicotinate 114 (160 g, 963 mmol) in t-BuOH (1.7 L) and acetone (560 mL) was added DMAP (2.38 g, 19.1 mmol) and di-t-butyl dicarbonate (420 g, 1.92 mol). The reaction was stirred at room temperature overnight. The solvent was removed and hexane/dichloromethane (2.5 L, 3:1) was added. The mixture was cooled to −20° C. for 2 h. The solid was collected by filtration and dried in vacuo to give 115 (300 g, 85% yield).

Step 3. Preparation of tert-butyl 5-(hydroxymethyl)pyridin-2-ylcarbamate (116)

To a stirred solution of ethyl 6-(bis(tert-butoxycarbonyl)amino)nicotinate 115 (300 g, 819 mmol) in THF (1.2 L) was added LiAlH₄ (57.6 g, 1.51 mol) in THF (3 L) over a period of 30 min at 0° C. The reaction mixture was stirred for 6 h, and H₂O (30.0 ml) and 10% NaOH solution (60.0 mL) were added. The solids were removed by filtration and the filtrate was dried (Na₂SO₄) and concentrated. The crude residue was purified by flash chromatography (CH₂Cl₂; MeOH=40:1) to give 116 (85.0 g, 46% yield).

Step 4. Preparation of tert-butyl 5-(chloromethyl)pyridin-2-ylcarbamate (117)

To a solution of tert-butyl 5-(hydroxymethyl)pyridin-2-ylcarbamate 116 (85.0 g, 379 mmol) and diisopropylethylamine (296 g, 2.27 mol) in THF (850 mL) was added methanesulfonyl chloride (130 g, 1.14 mol) over a period of 30 min at 0° C. The mixture was stirred for 12 h at room temperature then washed with H₂O (2×100 mL) and dried over Na₂SO₄. The mixture was concentrated and the crude residue was purified by flash chromatography (petroleum ether: ethyl acetate=10:1) to give 117 (30 g, 63% yield).

Step 5. Preparation of (S)-tert-butyl 5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-ylcarbamate (119)

A mixture of tert-butyl 5-(chloromethyl)pyridin-2-ylcarbamate 117(9.5 g, 39.1 mmol), (S)-3-fluoropyrrolidine 118 (4.19 g, 47.0 mmol), potassium carbonate (16.2 g, 117 mmol) and sodium iodide (0.586 g, 3.91 mmol) in DMF (150 mL) was stirred at 60° C. for 2 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo. H₂O (250 mL) was added and the resulting solid was collected by filtration, rinsed with H₂O and dried to give 119 (7.00 g, 61% yield).

Step 6. Preparation of (S)-5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine (120)

To a solution of (S)-tert-butyl 5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-ylcarbamate 119 (7.00 g, 23.7 mmol) in dichloromethane (70 mL) was added TFA (15.5 g, 142 mmol). The mixture was stirred for 12 h at room temperature. The solvent was removed in vacuo and sat. aq Na₂CO₃ was added. The mixture was extracted with dichloromethane, dried (MgSO₄) and concentrated to give 120 (4.50 g, 97% yield).

Example 47 Synthesis of 5-(morpholinomethyl)pyridin-3-amine 121

Compound 121 was prepared by a method similar to that reported for (S)-5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine 120.

Example 48 Synthesis of 6-(morpholinomethyl)pyridin-3-amine 122

Compound 122 was prepared by a method similar to that reported for (S)-5-((3-fluoropyrrolidin-1-yl)methyl)pyridin-2-amine 120.

Example 49 Synthesis of 2-(morpholinomethyl)pyrimidin-4-amine 127 Step 1. Preparation of 2-chloroacetimidamide dihydrochloride (124)

2-chloroacetonitrile 123 (300 g, 4.0 mol) was added to a solution of sodium (10.0 g, 0.43 mol) in methanol (1000 mL) keeping the temperature below 20° C. The mixture was stirred at room temperature for 2 h. NH₄Cl (234 g, 4.37 mol) was added in 5 batches and stirring continued for another 2 h. The solvent was removed to give 124 (525 g, 79% yield) which was used directly for next step without further purification.

Step 2. Preparation of 2-(chloromethyl)pyrimidin-4-amine (126)

A solution of 2-chloroacetimidamide dihydrochloride 124 (250.0 g, 1.51 mol), 2-chloroacrylonitrile 125 (171 g, 1.95 mol) and triethylamine (490 g, 4.8 mol) in anhydrous ethanol (600 mL) was heated at reflux for 30 min. The solvent was removed in vacuo and the residue was purified by flash chromatography (dichloromethane:methanol=30:1) to give 126 (39.0 g, 18% yield).

Step 3. Preparation of 2-(morpholinomethyl)pyrimidin-4-amine (127)

A solution of 2-(chloromethyl)pyrimidin-4-amine 126 (30.0 g, 209 mmol), morpholine 63 (23.7 g, 272 mmol) and triethylamine (42.3 g, 418 mmol) in anhydrous ethanol (250 mL) was heated at reflux for 16 h. The solvent was removed in vacuo and methanol (400 mL), H₂O (100 mL) and sodium bicarbonate (25.0 g) were added. Stirring was continued for 30 min. The mixture was concentrated and purified by flash chromatography (dichloromethane: methanol: triethylamine=100:8:0.5) to give 127 (25.0 g, 62% yield).

Example 50 Synthesis of tert-butyl 4-((4-aminopyrimidin-2-yl)methyl)piperazine-1-carboxylate 128

Compound 128 was prepared by a method similar to that reported for 2-(morpholinomethyl)pyrimidin-4-amine 127. The Boc protecting group could be removed after coupling with the appropriate carboxylic acid by treatment with TFA/CH₂Cl₂ for 12 h.

Example 51 Synthesis of 2-(pyrrolidin-1-ylmethyl)pyrimidin-4-amine 129

Compound 129 was prepared by a method similar to that reported for 2-(morpholinomethyl)pyrimidin-4-amine 127.

Example 52 Biological Activity

A mass spectrometry based assay was used to identify modulators of SIRT1 activity. The mass spectrometry based assay utilizes a peptide having 20 amino acid residues as follows: Ac-EE-K(biotin)-GQSTSSHSK(Ac)NleSTEG-K(5TMR)-EE-NH2 (SEQ ID NO: 1) wherein K(Ac) is an acetylated lysine residue and Nle is a norleucine. The peptide is labeled with the fluorophore 5TMR (excitation 540 nm/emission 580 nm) at the C-terminus. The sequence of the peptide substrate is based on p53 with several modifications. In addition, the methionine residue naturally present in the sequence was replaced with the norleucine because the methionine may be susceptible to oxidation during synthesis and purification.

The mass spectrometry assay is conducted as follows: 0.5 μM peptide substrate and 120 μM βNAD⁺ is incubated with 10 nM SIRT1 for 25 minutes at 25° C. in a reaction buffer (50 mM Tris-acetate pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 5 mM DTT, 0.05% BSA). Test compounds may be added to the reaction as described above. The SirT1 gene is cloned into a T7-promoter containing vector and transformed into BL21(DE3). After the 25 minute incubation with SIRT1, 10 μL of 10% formic acid is added to stop the reaction. Reactions are sealed and frozen for later mass spec analysis. Determination of the mass of the substrate peptide allows for precise determination of the degree of acetylation (i.e. starting material) as compared to deacetylated peptide (product).

A control for inhibition of sirtuin activity is conducted by adding 1 μL of 500 mM nicotinamide as a negative control at the start of the reaction (e.g., permits determination of maximum sirtuin inhibition). A control for activation of sirtuin activity is conducted using 10 nM of sirtuin protein, with 1 μL of DMSO in place of compound, to determine the amount of deacetylation of the substrate at a given timepoint within the linear range of the assay. This timepoint is the same as that used for test compounds and, within the linear range, the endpoint represents a change in velocity.

For the above assay, SIRT1 protein was expressed and purified as follows. The SirT1 gene was cloned into a T7-promoter containing vector and transformed into BL21(DE3). The protein was expressed by induction with 1 mM IPTG as an N-terminal His-tag fusion protein at 18° C. overnight and harvested at 30,000×g. Cells were lysed with lysozyme in lysis buffer (50 mM Tris-HCl, 2 mM Tris[2-carboxyethyl]phosphine (TCEP), 10 μM ZnCl₂, 200 mM NaCl) and further treated with sonication for 10 min for complete lysis. The protein was purified over a Ni-NTA column (Amersham) and fractions containing pure protein were pooled, concentrated and run over a sizing column (Sephadex S200 26/60 global). The peak containing soluble protein was collected and run on an Ion-exchange column (MonoQ). Gradient elution (200 mM-500 mM NaCl) yielded pure protein. This protein was concentrated and dialyzed against dialysis buffer (20 mM Tris-HCl, 2 mM TCEP) overnight. The protein was aliquoted and frozen at −80° C. until further use.

Sirtuin modulating compounds that activated SIRT1 were identified using the assay described above and are shown below in Tables 1 and 2. The EC_(1.5) values for the activating compounds are represented by A (EC_(1.5)<1.0 μM), B (EC_(1.5) 1-25 μM), C (EC_(1.5)>25 μM). The percent maximum fold activation is represented by A (Fold activation>200%) or B (Fold Activation<200%).

TABLE 1 Compounds wherein W¹ is —N═, and W² is N. Compd EC1.5 % Fold No [M + H]+ Structure μM Act. 200 447

B B 201 431

A B 202 426

A A 203 447

C B 204 442

A A 205 431

B B 206 425

A A 207 441

A A 208 425

C B 209 442

B A 210 357

B B 211 441

C B 212 425

B A 213 441

B A 214 349

C B 215 435

C B 216 442

C B 217 442

B A 218 411

A A 219 426

A A 220 484

A A 221 435

B A 222 435

B A 223 358

C B 224 335

C B 225 364

C B 226 378

B B 227 361

C B 228 361

B B 229 431

B B 230 429

A A 231 412

C B 232 440

A A 233 454

A A 234 468

A A 235 509

B A 236 428

B A 237 425

B B 238 428

B A 239 425

A A 240 425

B A 241 426

C B 242 391

B B 243 391

B B 244 391

C B 245 426

A A 246 428

A A 247 428

B B 248 428

A A 249 426

A A 250 415

C B 251 426

B B 252 442

C B 253 382

C B 254 382

C B 255 358

C B 256 358

C B 257 359

C B 258 359

C B 259 359

C B 260 359

C B 261 361

C B 262 361

C B 263 361

C B 264 361

C B 265 425

B A 266 425

B A 267 454

A A 268 428

C B 269 439

B B 270 453

C B 271 467

B B 272 508

B B 273 514

A A 274 442

A A 275 440

C B 276 440

B B 277 359

C B 278 359

B B 279 359

C B 280 360

C B 281 360

C B 282 360

C B 283 365

C B 284 379

B A 285 362

B B 286 514

A A 287 393

B A 288 362

C B 289 362

C B 290 362

C B 291 362

C B 292 362

C B 293 510

A A 294 460

A A 295 442

C B 296 445

B B 297 488

A A 298 459

A A 299 432

B A 300 524

B A 301 524

B A 302 523

A A 303 426

B A 304 510

A A 305 429

B A 306 453

A A 307 525

B A 308 530

B A 309 530

A A 310 408

A A 311 394

C B 312 424

A A 313 410

A A 314 408

A A 315 424

A A 316 410

C B 317 412

A A 318 428

A A 319 393

C B 320 427

A A 321 411

B A 322 407

B A 323 407

C B 324 393

A A 325 409

C B 326 423

A A 327 423

B A 328 409

B A 329 394

A A 330 509

A A 331 431

B B 332 524

A A 333 508

A A 334 515

B A 335 515

A A 336 511

A A 337 493

C B 338 523

A A 339 431

C B 340 445

C B 341 510

A A 342 494

A A 343 445

C B 344 524

A A 345 526

A A 346 526

A A 347 524

A A 348 523

A A 349 494

A A 350 494

B B 351 508

A A 352 526

A A 353 515

A A 354 516

A A 355 516

A A 356 515

A A 357 514

A A 358 514

B A 359 509

B A 360 514

A A 361 461

C B 362 461

C B 363 514

A A 364 507

B A 365 445

B A 366 529

A A

In certain embodiments, the compound of this invention is selected from any one of Compounds 201, 202, 204, 206, 207, 218, 219, 220, 230, 232, 233, 234, 239, 245, 246, 248, 249, 267, 273, 274, 286, 293, 294, 297, 298, 302, 304, 306, 309, 310, 312, 313, 314, 315, 317, 318, 320, 324, 326, 329, 330, 332, 333, 335, 336, 338, 341, 342, 344, 345, 346, 347, 348, 349, 351, 352, 353, 354, 355, 356, 357, 360, 363, and 366 from Table 1. In one aspect, the compound is selected from any one of Compounds 201, 202, 204, 218, 220, 230, 239, 245, 248, 249, 274, 286, 293, 309, 310, 312, 313, 314, 315, 317, 318, 329, 330, 332, 333, 335, 336, 341, 344, 345, 346, 349, 352, 354, 355, 357, and 360.

TABLE 2 Compounds wherein W¹ is N and W² is C. Compd EC1.5 % Fold No [M + H]+ Structure μM Act. 400 445

A A 401 410

A A 402 410

A A 403 411

A A 404 416

A A 405 416

A A 406 414

A A 407 427

A A 408 410

A A 409 411

A A 410 427

A A 411 416

A A 412 417

B A 413 417

B B 414 442

B A 415 411

A A 416 416

A A 417 411

A A 418 411

A A 419 410

A A 420 413

A A 421 411

B A 422 440

A A 423 471

A A 424 428

A A 425 495

A A 426 382

A A 427 416

A A 428 414

A A 429 432

A A 430 432

A A 431 398

A A 432 393

A A 433 382

A A 434 377

A B 435 398

B A 436 414

A A 437 410

A A 438 392

B A 439 426

A A 440 408

B A 441 376

B A 442 427

A A 443 408

A A 444 416

A A 445 411

A A 446 382

B A 447 377

A A 448 509

A A 449 417

A A

In certain embodiments, the compound of this invention is selected from any one of Compounds 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 415, 416, 417, 418, 419, 420, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 436, 437, 439, 442, 443, 444, 445, 447, 448, and 449 from Table 2. In one aspect, the compound of this invention is selected from any one of Compounds 400, 401, 402, 403, 404, 405, 406, 408, 409, 410, 415, 417, 418, 420, 423, 424, 425, 428, 431, 432, 434, 442, 443, 445, and 448.

EQUIVALENTS

The present invention provides among other things sirtuin-activating compounds and methods of use thereof. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). 

The invention claimed is:
 1. A compound of Formula (IIA) or Formula (IIB):

wherein: each R⁹ is independently selected from hydrogen, halo, —C≡N, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, C₁-C₄ alkyl, —S—(C₁-C₄) alkyl, C₃-C₇ cycloalkyl, —(C₁-C₂) alkyl-N(R¹³)(R¹³), —O—CH₂CH(OH)CH₂OH, —O—(C₁-C₃) alkyl-N(R¹³)(R¹³), and —N(R¹³)(R¹³); R¹¹ is selected from a carbocycle and a heterocycle; wherein: R¹¹ is optionally substituted with one to two substituents independently selected from halo, —C≡N, C₁-C₄ alkyl, ≡O, C₃-C₇ cycloalkyl, fluoro-substituted C₁-C₄ alkyl, —O—R¹³, —S—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), and —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³); and when R¹¹ is phenyl, R¹¹ is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, —O-(saturated heterocycle), fluoro-substituted-O-(saturated heterocycle), or C₁-C₄ alkyl-substituted —O-(saturated heterocycle); wherein: each R¹³ is independently selected from hydrogen, and —C₁-C₄ alkyl; or two R¹³ are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered saturated heterocycle optionally comprising one additional heteroatom selected from N(R¹³), S, S(═O), S(═O)₂, and O, wherein when R¹³ is alkyl, the alkyl is optionally substituted with one or more —OH, fluoro, —NH₂, —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂; and when two R¹³ are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered saturated heterocycle, the saturated heterocycle is optionally substituted at a carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂, and optionally substituted at any substitutable nitrogen atom with —C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, or —(CH₂)₂—O—CH₃; R¹² is selected from a carbocycle and a heterocycle other than tetrazolyl; wherein: R¹² is optionally substituted with one to two substituents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹³, —S—R¹³, —S(O)—R¹³, —S(O)₂—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³), —O-phenyl, phenyl, and a second heterocycle, and when R¹² is phenyl, R¹² is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, or —O-(saturated heterocycle); wherein: any phenyl, second heterocycle or saturated heterocycle portion of a substituent of R¹² is optionally substituted with halo; —C≡N; C₁-C₄ alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, —NH—(C₁-C₄) alkyl and —N—(C₁-C₄)₂ alkyl; R¹⁴ is selected from hydrogen, C₁-C₄ alkyl, C₁-C₄ fluoro-substituted alkyl, C₁-C₄ alkyl-N(R¹³)(R¹³), C₁-C₄ alkyl-C(O)—N(R¹³)(R¹³), C₁-C₄ alkyl-O—R¹³, and C₁-C₄ alkyl-NR¹³—C(O)R¹³; X¹ is selected from —NH—C(═O)-†, —C(═O)—NH-†, or —NH—S(═O)₂-†; wherein: † represents where X¹ is bound to R¹¹; and each R¹⁵ and R¹⁶ is independently selected from hydrogen, C₁-C₄ alkyl, —CF₃ and (C₁-C₃ alkyl)-CF₃; or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein X¹ is selected from —NH—C(O)-† or —C(O)—NH-†.
 3. The compound of claim 1, wherein R¹¹ is selected from:

wherein R¹¹ is optionally substituted with one or two substituents independently selected from halo, C₁-C₄ alkyl, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), ═O, —N(R¹³)(R¹³), and —O—R¹³.
 4. The compound of claim 3, wherein R¹¹ is selected from:


5. The compound of claim 4, wherein R¹¹ is selected from


6. The compound of claim 1, wherein R¹² is selected from:

wherein: R¹² is optionally substituted with one or two groups independently selected from halo, C₁-C₄ alkyl, —(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴), C₁-C₂ fluoro-substituted alkyl, —O—R¹⁴, —SO₂—R¹⁴, —N(R¹⁴)(R¹⁴), and —O—(C₁-C₄ alkyl)-N(R¹⁴)(R¹⁴).
 7. The compound of claim 6, wherein R¹² is selected from:


8. The compound of claim 7, wherein R¹² is selected from


9. A compound which is: Compound No Structure 201

202

204

206

207

218

219

220

230

232

233

234

239

245

246

248

249

267

273

274

286

293

294

297

298

302

304

306

309

310

312

313

314

315

317

318

320

324

326

329

330

332

333

335

336

338

341

342

344

345

346

347

348

349

351

352

353

354

355

356

357

360

363

366

400

401

402

403

404

405

406

407

408

409

410

411

415

416

417

418

419

420

422

423

424

425

426

427

428

429

430

431

432

433

434

436

437

439

442

443

444

445

447

448

449

or a pharmaceutically acceptable salt thereof.
 10. A compound which is selected from any one of the Compounds from the following table: Compound No Structure 400

401

402

403

404

405

406

407

408

409

410

411

415

416

417

418

419

420

422

423

424

425

426

427

428

429

430

431

432

433

434

436

437

439

442

443

444

445

447

448

449

or a pharmaceutically acceptable salt thereof.
 11. A compound which is selected from the following table: Compound No Structure 400

401

402

403

404

405

406

408

409

410

415

417

418

420

423

424

425

428

431

432

434

442

443

445

448

or a pharmaceutically acceptable salt thereof.
 12. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipient, carrier or diluent.
 13. A method for treating insulin resistance, a metabolic syndrome, diabetes, or complications thereof, or for increasing insulin sensitivity in a subject, comprising administering a pharmaceutical composition of claim 12 to a subject in need thereof.
 14. A compound of Formula (IIB):

wherein: each R⁹ is independently selected from hydrogen, halo, —C≡N, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, C₁-C₄ alkyl, —S—(C₁-C₄) alkyl, C₃-C₇ cycloalkyl, —(C₁-C₂) alkyl-N(R¹³)(R¹³), —O—CH₂CH(OH)CH₂OH, —O—(C₁-C₃) alkyl-N(R¹³)(R¹³), and —N(R¹³)(R¹³); R¹¹ is selected from a carbocycle and a heterocycle; wherein: R¹¹ is optionally substituted with one to two substituents independently selected from halo, —C≡N, C₁-C₄ alkyl, ═O, C₃-C₇ cycloalkyl, fluoro-substituted C₁-C₄ alkyl, —O—R¹³, —S—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), and —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³)—; and when R¹¹ is phenyl, R¹¹ is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, fluoro-substituted 3,4-ethylenedioxy, —O-(saturated heterocycle), fluoro-substituted-O-(saturated heterocycle), or C₁-C₄ alkyl-substituted —O-(saturated heterocycle); wherein each R¹³ is independently selected from hydrogen, and —C₁-C₄ alkyl; or two R¹³ are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered saturated heterocycle optionally comprising one additional heteroatom selected from N(R¹³), S, S(═O), S(═O)₂, and O, wherein when R¹³ is alkyl, the alkyl is optionally substituted with one or more —OH, fluoro, —NH₂, —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂; and when two R¹³ are taken together with the nitrogen atom to which they are bound to form a 4- to 8-membered saturated heterocycle, the saturated heterocycle is optionally substituted at a carbon atom with —OH, —C₁-C₄ alkyl, fluoro, —NH₂, —NH(C₁-C₄ alkyl), —N(C₁-C₄ alkyl)₂, —NH(CH₂CH₂OCH₃), or —N(CH₂CH₂OCH₃)₂, and optionally substituted at any substitutable nitrogen atom with —C₁-C₄ alkyl, fluoro-substituted C₁-C₄ alkyl, or —(CH₂)₂—O—CH₃; R¹² is selected from a carbocycle and a heterocycle other than tetrazolyl; wherein: R¹² is optionally substituted with one to two substituents independently selected from halo, —C≡N, C₁-C₄ alkyl, C₃-C₇ cycloalkyl, C₁-C₂ fluoro-substituted alkyl, —O—R¹³, —S—R¹³, —S(O)—R¹³, —S(O)₂—R¹³, —(C₁-C₄ alkyl)-N(R¹³)(R¹³), —N(R¹³)(R¹³), —O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —(C₁-C₄ alkyl)-O—(C₁-C₄ alkyl)-N(R¹³)(R¹³), —C(O)—N(R¹³)(R¹³), —(C₁-C₄ alkyl)-C(O)—N(R¹³)(R¹³), —O-phenyl, phenyl, and a second heterocycle, and when R¹² is phenyl, R¹² is also optionally substituted with 3,4-methylenedioxy, fluoro-substituted 3,4-methylenedioxy, 3,4-ethylenedioxy, or fluoro-substituted 3,4-ethylenedioxy, or —O-(saturated heterocycle); wherein: any phenyl, second heterocycle or saturated heterocycle portion of a substituent of R¹² is optionally substituted with halo; —C≡N; C₁-C₄ alkyl, fluoro-substituted C₁-C₂ alkyl, —O—(C₁-C₂) fluoro-substituted alkyl, —O—(C₁-C₄) alkyl, —S—(C₁-C₄) alkyl, —S—(C₁-C₂) fluoro-substituted alkyl, —NH—(C₁-C₄) alkyl and —N—(C₁-C₄)₂ alkyl; R¹⁴ is selected from hydrogen, C₁-C₄ alkyl, C₁-C₄ fluoro-substituted alkyl, C₁-C₄ alkyl-N(R¹³)(R¹³), C₁-C₄ alkyl-C(O)—N(R¹³)(R¹³), C₁-C₄ alkyl-O—R¹³, and C₁-C₄ alkyl-NR¹³—C(O)R¹³; X¹ is selected from —NH—C(═O)-†, —C(═O)—NH-†; wherein: † represents where X¹ is bound to R¹¹; and each R¹⁵ and R¹⁶ is independently selected from hydrogen, C₁-C₄ alkyl, —CF₃ and (C₁-C₃ alkyl)-CF₃; or a pharmaceutically acceptable salt thereof.
 15. A compound which is: Compd No Structure 200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

or a pharmaceutically acceptable salt thereof.
 16. A compound which is: Compd No Structure 361

362

or a pharmaceutically acceptable salt thereof. 